Measuring B-lymphocyte responses in human health and primary immunodeficiency
AuthorTempany, Jessica Catherine
Document TypePhD thesis
Access StatusThis item is embargoed and will be available on 2021-11-20.
© 2019 Jessica Catherine Tempany
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.
KeywordsImmunology; Humoral immunity; Immunodeficiency; Primary immunodeficiency; Common variable immunodeficiency; B cells; B cell function; In vitro assay
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- Medical Biology - Theses