An investigation of in vivo antigen-specific B cell responses

Abstract

To optimize the initial wave of antibody production against T-dependent antigens, primary B cell clones with the highest antigen affinity are selected to generate the largest extrafollicular plasmablast (PB) responses. The mechanism behind this remains undefined, primarily due to the difficulty of analysing low-frequency antigen-specific B cells during the earliest phases of the immune response when key differentiation decisions are made. In this thesis a high resolution in vivo mouse model, namely the adoptive transfer and challenge of anti-hen egg lysozyme (SWHEL) B cells, was used to characterize in detail the first six days of a T-dependent B cell response and to identify the steps at which initial antigen affinity has a major impact. Antigen-specific B cells proliferated within splenic follicles from days 1.0-3.0 before undergoing a dynamic phase of multi-lineage differentiation (days 3.0-4.0) that generated switched and unswitched populations of germinal centre (GC) B cells, early memory B cells, and extrafollicular PBs. Differences in antigen affinity of >50-fold did not markedly affect the early stages of the response, including the differentiation and extrafollicular migration of PBs. However, high affinity PBs underwent significantly greater expansion within the splenic bridging channels and red pulp, due to both increased proliferation and decreased apoptosis. This thesis also describes an adaptation to the adoptive transfer strategy utilizing the SWHEL adoptive transfer model that enables the investigation of tolerance mechanisms that exist in the periphery. Within the GC, the random nature of somatic hypermutation gives rise to the possibility that B cells may generate self-reactive specificities. A description is provided of the design and production of a novel in vivo mouse model (HEL4X transgenic) engineered to study autoreactivity acquired during the GC reaction. Preliminary experiments are also described which demonstrated that upon encounter of a ubiquitously-expressed cross-reactive autoantigen, high- but not low-affinity autoreactive B cell clones generated in the GC undergo rapid deletion. This model provides an ideal system for defining the mechanisms that mediate inactivation of autoreactive B cells in the GC and how these cells may escape and differentiate into plasma cells that may potentially produce pathogenic autoantibody responses.