B cell differentiation in the germinal center reaction and the resulting memory B cell compartment in humans

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The germinal center (GC) reaction is the central process of T cell dependent humoral immune responses and is essential for affinity maturation of antibodies and the generation of long-lived memory B cells and plasma cells. In this process, antigen-activated GC B cells undergo cycles of proliferation and selection, coupled with somatic hypermutation of immunoglobulin V (IGV) genes and class-switch recombination. Highly dynamic differentiation processes take place, in which it is decided whether a GC B cell upon selection is eliminated by apoptosis, undergoes a new round of proliferation and mutation in the GC, or differentiates into a memory or plasma cell and exits the GC. We have first insights into these processes, but still lack a comprehensive understanding of the diversity of GC B cells and the distinctness of their differentiation processes. We recently identified CD30+ GC B cells as the subset returning to the proliferative state. Our first aim is to characterize the heterogeneity of GC B cells and the dynamics of their differentiation processes by single cell RNA-sequencing, combined with IGV gene sequencing of the single cells. We will test specific hypotheses with this approach. There are also still major uncertainties regarding the generation of memory B cells from positively selected GC B cells and the complexity of the resulting memory B cell compartment. Recently, we performed an initial high throughput sequencing (HTS) analysis of rearranged IGV genes of three subsets of human memory B cells, revealing that IgM and IgG memory B cells can derive from common GC B cell clones. As this preliminary approach was limited in terms of cell numbers and memory B cell subsets, the second major aim of our proposal is to characterize the complexity of the human memory B cell compartment comprehensively by HTS of IGV genes from seven memory B cell subsets. By using leukapheresis samples for this, we can reach an unprecedented depth and novel quality of analysis, which is critical for a reliable determination of the size and complexity of memory B cell clones. Through repeated analysis of the donors after two years we can furthermore study the stability and longevity of memory B cell clones.