An inquiry into the regulation of positive selection and differentiation in the germinal centre response

Abstract

In response to antigenic challenge, the production of high-affinity, antibody secreting plasma cells (PCs) and memory B cells (MBCs) by germinal centres (GCs) confers immunological protection against re-infection by the same pathogen and unpins the efficacy of nearly all vaccines currently in human use. Here, the high-resolution SWHEL B cell transgenic model is combined with a number of gene-knockout and antibody-interference perturbations in order to investigate the role of antigen affinity versus T-cell help in the regulation of positive selection and differentiation in the GC response. SWHEL B cells were adoptively transferred into CD45-congenic recipients and challenged with a low-affinity antigen (HEL3X conjugated to sheep red blood cells). Affinity maturation was observed and measured by flow cytometry, and PC differentiation tracked using a Blimp1-GFP reporter mouse. The impact of blocking access to antigen or T-cell help was investigated using depleting or blocking antibodies. As expected, blocking access to antigen during the GC response impaired affinity maturation and almost completely abolished the production of PCs. In stark contrast, and unexpectedly, depleting CD4+ T cells accelerated affinity maturation and had no impact on the production of high-affinity PCs, but rather caused PCs to become trapped in the GC light zone. Using a separate murine system, the surprising observation was made that low-affinity GC B cells present more peptide:MHC-II on their surface compared to high-affinity GC B cells. Taken together, these data are consistent with a model whereby direct competition for antigen-dependent signals, not limiting T-cell help, drives positive selection and differentiation of high-affinity GC B cells into PCs. Additionally, the chemokine receptor CCR6 was found to identify the subpopulation of GC B cells fated to differentiate into MBCs. This CCR6-expressing GC B-cell population was characterised in detail in two murine systems, and a corresponding population of CCR6-expressing MBC precursors was also noted in human tonsils, resolving the longstanding question of how to identify MBC precursors in the GC. Finally, a microarray was performed on human tonsil GCs in order to identify the genes that are most differentially expressed at the earliest stages of commitment to an MBC fate.