Defining B cell tolerance checkpoints and their modulation by immunotherapy

Abstract

B cells that recognise self-antigens are thought to be deleted from the emerging repertoire and those which escape deletion are instead locked in an anergic state. Autoimmunity develops when this process breaks down. This paradigm was established almost three decades ago in mouse models where all B cells recognise a single model antigen. Since then, very little about B cell tolerance has been learned from polyclonal settings where mice express a normal endogenous repertoire. This is because we have lacked tools to dissect B cell tolerance within a physiological repertoire in fine detail. This thesis aimed to understand polyclonal B cell tolerance and ways to modulate it. To study B cell tolerance, I took advantage of mouse models that express membranebound ovalbumin (OVA) as a model self-antigen. Three models were used where OVA was expressed ubiquitously, by MHC class II-expressing cells, or by CD11c+ dendritic cells. I generated fluorescent OVA tetramers that could probe for OVA-specific B cells in a polyclonal repertoire in both naïve mice and following immunization. Surprisingly, at steady-state, self-reactive OVA-specific B cells populated OVA-expressing mice in a manner that suggested deletion during B cell development does not occur. Instead, the large number of self-reactive B cells in the peripheral repertoire are maintained as anergic cells. This anergic state largely prevented responses to self-antigen when mice were immunized with OVA in the presence of different immunogenic stimuli. Of the small number of self-reactive cells that became activated in OVA-expressing mice, these were rapidly deleted from germinal centres and OVA-specific antibody production was transient. Providing a source of T cell help, either cognate or from bystander T cells, failed to reverse anergy, indicating that anergy was B cell-intrinsic and not as a consequence of T cell tolerance. Collectively, these studies demonstrated checkpoints that maintain robust peripheral B cell tolerance in a polyclonal repertoire. As a way to modify an existing repertoire, I transplanted OVA-encoding bone marrow and tested whether OVA-specific responsiveness could be modulated following engraftment. OVA-specific B cells could be edited from the repertoire and rendered anergic by OVA expression as a neo-self-antigen, demonstrating an approach that could harness peripheral tolerance to control B cells. These studies refine our current understanding of B cell tolerance. Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, financial support and any other original research work used or reported in my th...