Germinal centers (GCs) form transiently in secondary lymphoid organs following immunization or infection and are central in providing humoral immunity. Within the GC, antigen-specific B lymphocytes express activation-induced cytidine deaminase (AID), inducing somatic hypermutation (SMH) and class switch recombination (CSR). These processes randomly mutate the immunoglobulin locus and switch antibody isotype, respectively. Competitive selection ensues among mutant GC B cells, with high affinity B cells undergoing selective expansion and differentiating into memory or plasma cells. Although the GC has long been studied, the events controlling selection of high affinity B cells remain obscure. The GC is subdivided into two anatomically and functionally distinct regions, the light (LZ) and dark zones (DZ). In the LZ, B cells bind antigen, retained as immune complexes on follicular dendritic cells (FDCs), in proportion to their affinity and process it for presentation to CD4+ T cells as peptide bound to major histocompatibility complex II (pMHCII). In this manner, high affinity GC B cells are selected based on pMHCII and T cell help, but the mechanisms by which this occurs are not known. Furthermore, a direct role for the B cell receptor (BCR) in GC selection, in addition to endocytosing antigen, has been suggested but never elucidated. This issue is further complicated by ongoing isotype switching in the GC, which changes the BCR signaling capacity. This proposal seeks to analyze how pMHCII levels and isotype-specific BCR signal transduction regulate GC B cell selection mechanisms. Toward the first goal, an antigen delivery system will be used that can modulate pMHCII levels on a subset of GC B cells in a temporally controlled manner. Toward the second goal, novel mice will be generated in which the isotype of the BCR can be switched through cre recombinase, independently of antibody affinity and somatic hypermutation. These problems, central to GC biology and the humoral immune response, will be addressed through a combination of mouse molecular genetics, traditional immunology techniques, flow cytometry, and multiphoton intravital imaging of live animals undergoing GC responses.
Vaccines have been among the most important public health measures instituted, as a result of immunological research. Yet for many pathogens, no adequately protective vaccine exists. The research proposed here will analyze the mechanisms by which high affinity antibodies are effectively produced through the germinal center reaction, with potential implications for novel vaccine development against such pathogens.