Molecular control of germinal center selection and affinity maturation

PROJECT SUMMARY Generation of high affinity antibodies in germinal centers (GCs) is a fundamental immunological process that provides protection against infection. Antibody affinity maturation follows a prototypical Darwinian evolutionary framework, in which rare GC B cells that acquire affinity-increasing mutations in the antigen-binding portions of their immunoglobulin genes selectively undergo massive proliferation, leading to their expansion within the GC population at the expense of lower-affinity clones. Through iterative rounds of selection and expansion, GC B cells increase their affinity toward a particular pathogen or antigen, while being exported as memory or plasma cells and contributing to the affinity of serum antibody over time. GC B cells are among the fastest dividing mammalian cells and are uniquely equipped with a distinct cell cycle program that allows them to divide every 4-6 hours. At its extreme, this robust expansion program can lead to clonal bursts, in which a single B cell can take over a 2,000-cell GC in just a few days. Interestingly, GC B cells can enter S phase in the apparent absence of mitogen, as if by “inertia,” hence their rapid mode of cell division is termed “inertial cycling”. Notably, many of the key drivers of GC-derived B cell lymphoma, such as cyclin D3, are critical for triggering and sustaining strong cell cycles in GC B cells. Despite its importance in the expansion of high-affinity B cell clones and development of GC-derived lymphomas, the precise cellular dynamics and molecular pathways that underlie inertial cycling of GC B cells remain poorly understood. This gap in our understanding is primarily due to a lack of tools with the necessary spatial and temporal sensitivity to resolve GC B cell cycles in vivo. The main goal of this research proposal is to define the cellular and molecular processes that underly the unique cell cycle programs of GC B cells. To achieve this, we will characterize the spatiotemporal dynamics of inertial cycling at a single cell level with intravital two-photon microscopy and fluorescent activity sensors (Aim 1); and we will determine the principles that coordinate inertial cell cycling and mutability of B cell receptors (Aim 2). Completion of the proposed Aims will provide a comprehensive understanding of the specialized inertial mode of GC B cell cycling and will contribute new insights into the fundamental mechanistic basis of how GCs select for high affinity B cells. In addition, the findings from this proposed Aims will better our understanding of GC- derived lymphomas, where inertial cycling becomes coopted for malignant transformation.