New perspectives on the evolution of B-lymphocytes in germinal centers

Central to the humoral immune response is the commonly observed improvement of antibody affinity over time, a phenomenon referred to as affinity maturation. Affinity maturation takes place in so-called germinal centers (GC) that are transiently formed in secondary lymphoid tissues (e.g. spleen) following immunization with T cell-dependent antigens. Within GC, B lymphocytes are subjected to a micro-evolutionary process that includes multiple rounds of diversification of their B cell receptors (BCRs) by somatic hypermutation (SHM) and subsequent selection of those B cells showing improved binding characteristics towards the antigen. However, despite recent advances in defining the mechanisms contributing to diversification of B lymphocytes within GC, the dynamics, mechanisms and forces of their selection are poorly understood. The current thesis aims at providing new insights into the evolution of B cells within GC by assessing the overall GC growth behavior (Chapter 4), by documenting changes of the cellular composition of GC over time (Chapter 5) and by, for the first time, specifying the kinetics (Chapter 5) and sequence properties (Chapter 6) of negative selection occurring within GC. The results obtained during cross-sectional evaluation of spleen sections revealed a robust average growth kinetics of GC, that was, however, based on a notable size distribution of GC within individual sections. This could not be explained by random sectioning of GC. However, simulations of random sectioning of hypothetical ensemble kinetics have shown that the experimentally recorded cross-sectional profile of GC growth is consistent with others than the accepted notion. To solve this problem, GC growth was subsequently addressed by three-dimensional volumetric evaluation of splenic GC, that finally contradicted the accepted notion by revealing non-synchronized formation and growth of GC as characterized by ongoing new formation and marked, broad volume distributions of GC. According to these findings, a revised model of affinity maturation was proposed that involves recirculation of GC emigrant B cells and a multilevel selection strategy (intercalation of local and global selection). It was further shown that the kinetics of GC T cells and macrophages follow the overall growth kinetics of GC. However, whereas T cells populated B cell zones not until antigenic challenge, macrophages already represented a constituent cell population of primary B cell follicles. Noteworthy, the number of macrophages was found to strictly correlate with the underlying size of B cell zones. In this context, an acquired imbalance due to phagocytosis-induced macrophage death was further shown to be associated with disease progression in the NZB/W autoimmune mouse model of systemic lupus erythematosus (SLE). Furthermore, costaining of macrophages and apoptotic cells revealed that cell death occurs throughout GC, thereby indicating that selection takes place in both, dark and light zones of GC. The establishment of a reliable method for recovery of primary BCR sequences of phagocytosed GC B cells has proven to be powerful, in that it provides new insights into the selection process of GC B cells by, for the first time, allowing for access to yet unexplored negatively selected GC B cells. In this context, the traditionally proposed molecular signatures as for instance the distribution of mutations within framework (FR) and complementary determining regions (CDRs), ratios of replacement (R) to silent mutations (S) and the incidence of key mutations were all shown to fail to demarcate winners and losers of selection. The obtained results indicated that the survival fate of GC B cells is governed by the cumulative effect of replacement mutations within CDRs, though, in which the as yet proposed positive effect of R mutations turned out to be overemphasized. By contrast, R mutations in CDRs turned out to possess marked deleterious effects, thereby lending support to the proposition that survival of GC B cells is particularly driven by the absence of excess mutations within CDRs that have an adverse effect with respect to antigen binding. Moreover, the high interclonal diversity of phagocytosed B cell fractions demonstrated that interclonal competition is sustained throughout the GC response most probably incited by ongoing replenishment of the pool of interacting B cells. Finally, this finding closes the circle to the central asssumption of the revised model of affinity maturation, that is recirculation of GC emigrant B cells. In conclusion, the current thesis provides new insights into the evolution of B cells within GC by proposing: • non-synchronized GC formation and growth • a multilevel selection strategy (intercalation of local and global selection by recirculation of GC emigrant B cells) • a central role for macrophages in retaining germinal center B cell homeostasis and preventing autoimmunity • the failure of commonly supposed molecular signatures to demarcate positively and negatively selected B cells • survival fate of GC B cells is particularly driven by absence of excess mutations within CDRs that have an adverse effect with respect to antigen binding