Todd, Sarah Katrina; (2019) The role B cells in ANCA-associated vasculitis. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

B cells are central to the pathology of ANCA-associated vasculitis (AAV), a disease characterized by autoantibodies and effectively treated by rituximab. In addition to promoting inflammation, a subset of B cells act to suppress harmful autoimmune responses. The balance of effector and regulatory B cell subsets in AAV is not known. This study was conducted to assess the relative frequency of these subsets during different states of disease activity. B regulatory cells are reduced in AAV, whether defined as CD5+ or CD24high CD38high. There is an imbalance in B regulatory (Breg) and B memory (Bmem) cells, which represents a proinflammatory B cell profile. Bmem augment Th1 differentiation in vitro, whilst Breg do not. Breg positively correlate with IL-10 competent B cells and, inversely with TNFα production in B cells. The converse was observed with Bmem. There was no functional defect in Breg from AAV patients, assessed by effect on Th1 differentiation and IL-10 induction in vitro. The imbalance in Bmem and Breg was represented as ratio, calculated from the number of memory cells divided by number regulatory cells in flow cytometry gate (M:Rn). M:Rn has potential for to be used as a predictive measure of clinical outcome. This imbalance may contribute to the high rate of relapse observed in AAV. Higher ratios were observed in PR3-ANCA patients (who have greatest propensity to relapse), compared to MPO-ANCA patients with the same disease activity. Tolerant patients and those treated with rituximab, had low M:Rn. Rituximab resets the balance of effector and regulatory B cells in AAV, including a tolerogenic state. Detection of proinflammatory and inhibitory immune mediators in B cells by intracellular flow cytometry, warranted additional analysis to better define the cytokine profile. 15 analytes were measured in cell supernatants. IL-10, IL-6, IL-1β, TNFα, IL-17A, IFNγ and IL-2 were significantly induced in PBMC, stimulated for maximum IL-10 induction in B cells. The net effect of these supernatants was to limit T cell TNFα production in vitro, this was not impaired on blockade of IL-10 with an agonistic antibody. Highly activated CD4 cells were increased in AAV, whilst Treg were reduced. There was no relationship between Breg and Treg frequency in AAV. Patients had reduction in Breg or Treg, but not both (frequency lower than the mean of the healthy controls, minus 2 standard deviations). Further investigation was conducted in an MPO animal model of disease, in which passive transfer of antibodies or B cells are reported to be pathogenic. Limited disease was observed on cell transfer, despite equivalent autoantibody titres to MPO null, immunised mice. B cells or antibody alone are insufficient to cause disease, an initiating event is crucial with deposition of MPO on the microvasculature and tissue damage elicited by innate immune cells previously described. Without this inciting damage, adaptive immune ignorance is maintained and renal function unaffected.

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