To optimize the initial wave of Ab production against T-dependent Ags, primary B cell clones with the highest Ag affinity are selected to generate the largest extrafollicular plasmablast (PB) responses. The mechanism behind this remains undefined, primarily due to the difficulty of analyzing low frequency Ag-specific B cells during the earliest phases of the immune response when key differentiation decisions are made. In this study, a high resolution in vivo mouse model was used to characterize in detail the first 6 days of a T-dependent B cell response and to identify the steps at which initial Ag affinity has a major impact. Ag-specific B cells proliferated within splenic follicles from days 1.0 to 3.0 before undergoing a dynamic phase of multilineage differentiation (days 3.0–4.0) that generated switched and unswitched populations of germinal center B cells, early memory B cells, and extrafollicular PBs. PB differentiation was marked by synchronous up-regulation of CXCR4 and down-regulation of CXCR5 and the adoption of a unique BCRhigh phenotype by unswitched PBs. Differences in Ag 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. Extrafollicular PBs maintained class II MHC, but not IL-21R expression, and interacted directly with Ag-specific extrafollicular Th cells, suggesting that IL-21-independent T cell help may drive extrafollicular PB expansion in responses to foreign Ag.

Spleen-resident dendritic cell (DC) populations occupy sentinel positions for the capture and presentation of blood-borne antigens. Here we found a difference in expression of the chemotactic receptor EBI2 (GPR183) on splenic DC subsets and that EBI2 regulated the positioning and homeostasis of DCs in the spleen. EBI2 and its main ligand, 7α,25-OHC, were required for the generation of the splenic CD4(+) DC subset and the localization of DCs in bridging channels. Absence of EBI2 from DCs resulted in defects in both the activation of CD4(+) T cells and the induction of antibody responses. Regulated expression of EBI2 on DC populations is therefore critical for the generation and correct positioning of splenic DCs and the initiation of immune responses.

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Secondary diversification of the B cell repertoire by immunoglobulin gene somatic hypermutation in the germinal center (GC) is essential for providing the high-affinity antibody specificities required for long-term humoral immunity. While the risk to self-tolerance posed by inadvertent generation of self-reactive GC B cells has long been recognized, it has not previously been possible to identify such cells and study their fate. In the current study, self-reactive B cells generated de novo in the GC failed to survive when their target self-antigen was either expressed ubiquitously or specifically in cells proximal to the GC microenvironment. By contrast, GC B cells that recognized rare or tissue-specific self-antigens were not eliminated, and could instead undergo positive selection by cross-reactive foreign antigen and produce plasma cells secreting high-affinity autoantibodies. These findings demonstrate the incomplete nature of GC self-tolerance and may explain the frequent association of cross-reactive, organ-specific autoantibodies with postinfectious autoimmune disease.