Atherosclerosis is the most common cause of ischaemic heart disease and stroke which represent a global health concern responsible for significant levels of morbidity and mortality worldwide. It is a chronic inflammatory disease characterised by plaque build-up within arterial walls initiated by low density lipoprotein cholesterol. Elevated plasma LDL is a major risk factor for atherosclerosis and its immunogenic oxidation creates neo-epitopes that drive inflammatory immune responses underlying the pathogenesis of atherosclerosis.

The germinal centre is found within secondary lymphoid organs and is responsible for the production of high affinity antibody-producing plasma cells and long-lived memory B cells against antigens such as epitopes of oxidised LDL. Class-switched plasma cells and anti-oxLDL IgG antibodies have been detected within atherosclerotic plaques and sera of human and mice suggesting that the germinal centre response is pathogenically dysregulated in atherosclerosis.

The tamoxifen-inducible AID-CreERT2-Rosa-EYFP lineage tracing mouse model was used and crossed with the atherosclerosis-prone LDLr-deficient mouse model. This model inducibly and specifically labels germinal centre B cells and their progeny using tamoxifen allowing for tracking of atherosclerosis-specific B cell clones comprising germinal centre, memory, and plasma cells. LDLr-/- and LDLr+/- (‘WT’) mice were fed chow or high fat diet for up to 8 weeks and upon tamoxifen dosing (timing varied throughout studies) via intra-peritoneal injection or oral gavage, AID-expressing cells (germinal centre B cells) were fluorescently labelled with EYFP. Cells were analysed using flow cytometry and FlowJo software. All graphs and statistics were created using the GraphPad Prism 7.0.

In this thesis, I sought to characterise and understand the germinal centre B cell response in atherosclerosis using a lineage tracing mouse model. Firstly, the model was validated as EYFP cells were only detected after tamoxifen administration and were only present in germinal centre-derived cell populations. Furthermore, labelling efficiency was independent of time, diet, or genotype.

The germinal centre response was characterised in both WT and LDLr-/- mice demonstrating an time-dependent increase in germinal centre responses. Germinal centre reactions were exacerbated, greater in magnitude and degree of class-switching, by increased plasma cholesterol levels consequent of both HFD and knock out of the LDL receptor. The combination of HFD and LDLr-/- genotype, replicating atherosclerotic conditions, synergistically exacerbated germinal centre responses biased towards pathogenic cellular output due to elevated levels of class switching.

Due to the permanence of EYFP labelling, it was possible to track EYFP labelled cells over time and throughout the course of atherosclerosis. Tracking studies revealed that hyperlipidaemic conditions induced by HFD resulted in longer-lived EYFP clones which had increased propensity to undergo class-switching. Furthermore, there was a greater output of EYFP labelled memory and plasma cells.

To investigate the mechanism by which HFD induced greater germinal centre responses, interventions were conducted to block the impact of cholesterol and inflammation separately. Use of a cholesterol inhibitor drug and cholesterol free diet revealed that dietary cholesterol is key in driving the exacerbated germinal centre responses observed at early stages i.e., 4 weeks of atherosclerosis. Use of a cytokine inhibitor to dampen inflammation limited germinal centre responses at later stages of atherosclerosis i.e., 8 weeks, suggesting that inflammation plays an important role in germinal centre responses at this later timepoint. Thus, the model proposed is that dietary cholesterol is critical early on in atherosclerosis as disrupted lipid homeostasis results in autoimmune B cell responses with germinal centres primarily reacting to accumulated oxidised LDL while at later stages, the inflammation associated with atherosclerosis fuels exacerbated and pathogenic germinal centre responses.

To examine the role of IgG2c and its receptor FcgRIV in atherosclerosis, the impact of IgG2c antibody complexes was tested in vitro. IgG2c significantly enhanced TNF secretion, a marker of inflammation, in FcgRIV-expressing myeloid-derived monocytes. Thus, IgG2c has the potential to exacerbate inflammatory responses from plaque macrophages and dendritic cells.

In conclusion, the AID lineage tracing mouse model has been validated for use in the atherosclerosis setting. It has been characterised showing that germinal centre responses are exacerbated during the course of atherosclerosis and are skewed towards production of class- switched longer-lived B cell clones. Dietary cholesterol is the main driver of this pathogenic response in tandem with the inflammatory conditions caused by atherosclerosis. Germinal centre-derived IgG2c antibodies could play an important role in exacerbating inflammation within the plaque through plaque macrophages and dendritic cells. This thesis presents a model whereby atherogenic dyslipidaemia, as a result of elevated serum LDL levels, breaks down B cell tolerance. This results in autoimmune pathology characterised by exacerbated germinal centre responses skewed towards pathogenic antibody isotype clone production in response to LDL-induced inflammation. This provides an opportunity to target B cell-related atherosclerosis-specific responses therapeutically.