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

生发中心(GC)反应是T细胞依赖的体液免疫反应的中心过程，对于抗体的亲和力成熟和长寿命记忆B细胞和浆细胞的产生是必不可少的。在这个过程中，抗原激活的GC B细胞经历了增殖和选择周期，伴随着免疫球蛋白V(IGV)基因的体细胞超突变和类别切换重组。发生高度动态的分化过程，在这个过程中，决定一个GC B细胞在选择时是被凋亡消除，在GC中经历新一轮的增殖和突变，还是分化为记忆细胞或浆细胞并退出GC。我们对这些过程有了初步的了解，但对GC-B细胞的多样性及其分化过程的特殊性仍缺乏全面的了解。我们最近发现CD30+GC B细胞是恢复增殖状态的亚群。我们的第一个目标是通过单细胞RNA测序，结合单细胞的IGV基因测序，来表征GC B细胞的异质性及其分化过程的动力学。我们将用这种方法来检验具体的假设。关于从积极选择的GC B细胞产生记忆B细胞以及由此产生的记忆B细胞室的复杂性，仍存在重大不确定性。最近，我们对人类记忆B细胞的三个亚群的重排的IGV基因进行了初步的高通量测序(HTS)分析，发现IgM和IgG记忆B细胞可以来自常见的GC B细胞克隆。由于这一初步方法受到细胞数量和记忆B细胞亚群的限制，我们建议的第二个主要目的是通过从7个记忆B细胞亚群中提取IGV基因的HTS来综合表征人类记忆B细胞亚群的复杂性。通过使用白细胞分离样本，我们可以达到前所未有的深度和新的分析质量，这对于可靠地确定记忆B细胞克隆的大小和复杂性至关重要。通过两年后对供者的重复分析，可以进一步研究记忆B细胞克隆的稳定性和寿命。

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.