Medical Biology - Theses
Permanent URI for this collection
Search Results
1 - 4 of 4
-
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.
-
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.
-
ItemA quantitative analysis of B cell responses to specific antigen( 2008)Humoral immune responses arise when B lymphocytes respond to activation signals, enter mitosis and proliferate rapidly. Concurrent differentiation to antibody secreting and isotype switched effector cells is tightly linked to cell division, such that the degree of proliferation strongly influences the nature of the response that is mounted. Previous versions of a quantitative model of lymphocyte proliferation based on inherent variation in the time cells take to divide or die were able to accurately describe the entry of naïve, resting cells into division and subsequent population expansion. In the work described here, the model was tested and extended by investigating the proliferation cessation and population contraction phases of in vitro B cell responses. Experiments designed to assess the distribution of times to die of cells that had ceased proliferating revealed that the number of divisions achieved by individual cells is stochastically distributed in the population and varied in response to different stimuli. Both the concentration and duration of stimulation regulate the number of divisions undergone. A cell that stops dividing is described as having reached its division destiny. Further investigation revealed that cells reach a maximum division destiny even during repeated high-dose stimulation. This limit is dictated by cellular progression through divisions, and is not dependent on the survival capacity of the cells or time. Incorporation of division destiny in the quantitative model allows proliferation cessation to be described and the distribution of times to die after this point to be assessed. This extended model can describe the full course of in vitro lymphocyte proliferative responses to various different stimuli. (For complete abstract open document)
-
ItemResolving causes and consequences in a model of autoimmune disease( 2010)Antibody is an essential component of immunity that triggers a series of mechanisms to neutralise and clear pathogens. In a situation where the targets of antibody cannot be cleared, for instance when they are a normal part of the host, self-directed antibody activates these same mechanisms to drive continued destruction of host tissues. The production of antibody is therefore a critical point of regulation in the prevention of autoimmune disease. A sequence of control points exists that prevents B cells expressing self-reactive antibody from differentiating to form plasma cells. Once this differentiation step has occurred, a plasma cell can persist for years unimpeded. No current therapies target plasma cells and their capacity for persistence precludes any potential benefit of B cell depletion strategies. The factors required for plasma cell maintenance are inadequately characterised, and even less is known of the means by which plasma cell homeostasis is regulated. In systemic lupus erythematosus (SLE), an antibody-mediated autoimmune disease, plasma cells can accumulate and secrete excess antibody into the serum. When this antibody reacts against self-components it may trigger autoimmune disease. With time, the recruitment of auxiliary cell types into the disease process can compound disease development and promote the maturation of self-reactive plasma cells into high-affinity, isotype switched antibody-producing cells whose capacity for inflicting tissue damage is considerably amplified. This cyclic pattern of disease development confounds the study of underlying cell-intrinsic defects, obscuring causes of disease from those aspects of the phenotype that are a consequence of inflammatory processes occurring as part of disease progression. Using the Lyn-deficient mouse model of SLE, this thesis demonstrates that certain aspects of this antibody-mediated disease require the participation of T cells to establish destructive IgG-driven inflammatory processes. When the contribution of T cells is diminished, an IgA-mediated pathology is revealed that is entirely reliant on the presence of IL-6. Pharmacological inhibitors of IL-6 and concurrent blockade of T cell help may therefore be an effective strategy for preventing the production of pathogenic IgG and IgA autoantibodies in SLE patients. Occurring in parallel and independently of these factors is an accumulation of unswitched plasma cells in peripheral lymphoid organs of Lyn-deficient mice. These plasma cells appear to accumulate by virtue of their enhanced capacity for survival, which is a direct result of the loss of Lyn in these cells. Lyn normally limits STAT3 signalling following IL-6 stimulation of plasma cells and in its absence loss of negative regulation of STAT3 signalling may reduce the threshold for plasma cell survival by STAT3-dependent factors such as IL-6. This could allow plasma cells to accrue beyond normal homeostatic limits. An additional means of regulating the plasma cell response to IL-6 is the induction of SOCS3, which curtails the duration of STAT3 signalling. The consequences of deletion of SOCS3 in the B cell lineage were investigated, revealing a subtle role for this negative regulator during the antibody response to T cell-dependent antigen.