Spatiotemporal dynamics of calcium signaling and metabolism in germinal center B cells

作为体液免疫反应的亲和成熟位点，生发中心构成了次级淋巴器官中B细胞命运决定的重要检查点。生发中心提供区隔化的微环境，将抗原诱导的B细胞受体激活和亲和选择与体细胞超突变相关的B细胞分裂物理分离。两个生发中心室之间的细胞交换是高度动态的。此外，已知通过B细胞受体激活会引起B细胞代谢状态的深刻变化，并且已经提出触发“代谢时钟”，设置一个时间窗口，在此期间B细胞必须接收第二信号才能生存，例如T细胞的帮助。活体显微镜对生发中心动力学的理解有很大的帮助。然而，对于生发中心B细胞受体信号传导与代谢交叉的时空调控机制，以及对B细胞选择、增殖和分化的影响，目前尚不清楚。这部分是由于缺乏在体内单细胞水平上分析B细胞代谢和功能的工具。利用我们独特的成像工具，我们计划(i)确定生发中心B细胞体内细胞质钙与一般代谢活性之间的关系，（ii）评估NAD(P) h依赖的代谢途径，特别是产生活性氧的NADPH氧化酶，如何参与生发中心选择事件，以及（iii）确定不同生发中心区域内代谢微环境对B细胞活化的影响。本项目将首次提供生发中心B细胞体内钙信号传导与代谢活性的相关数据，评估生理条件下微环境对B细胞受体信号传导与代谢串音的影响。在研究单位内，我们将受益于提供B细胞代谢和信号传导知识的良好环境。这补充了我们在生发中心B细胞体内代谢和功能成像方面的专业知识，这些知识将提供给联盟成员。

As the site of affinity maturation for the humoral immune response, the germinal center constitutes an important checkpoint for B cell fate decisions in secondary lymphatic organs. Germinal centers provide compartmentalized microenvironments, physically separating antigen-induced B cell receptor activation and affinity-based selection from B cell division linked to somatic hypermutation. The cellular exchange between both germinal center compartments is highly dynamic. Furthermore, activation via the B cell receptor is known to induce profound changes in the metabolic status of B cells, and it has been proposed to trigger a “metabolic clock”, setting a time window during which B cells have to receive a second signal to survive, such as T cell help. Intravital microscopy has significantly contributed to our understanding of germinal center dynamics. However, neither the mechanism of spatiotemporal regulation at the intersection between B cell receptor signaling and metabolism in the germinal center, nor the consequences for B cell selection, proliferation and differentiation are well understood. This is partly due to the lack of tools for analyzing B cell metabolism and function at a single cell level in vivo. Using our unique imaging tools, we plan to (i) determine the relation between cytoplasmic calcium and general metabolic activity in germinal center B cells in vivo, (ii) assess how NAD(P)H-dependent metabolic pathways, particularly reactive oxygen species-producing NADPH oxidases, are involved in germinal center selection events, and (iii) define the impact of metabolic microenvironments within the different germinal center zones on B cell activation. This project will for the first time deliver correlative data on calcium signaling and metabolic activity of germinal center B cells in vivo, and assess the impact of the microenvironment on the crosstalk of B cell receptor signaling and metabolism under physiological conditions. Within the research unit, we will benefit from the excellent environment providing knowledge on B cell metabolism and signaling. This complements our expertise on in vivo metabolic and functional imaging of germinal center B cells, which will be made available to the members of the consortium.