Light- and dark-zone death dynamics Germinal centers (GCs) are areas within lymphoid organs where mature B cells expand and differentiate during normal immune responses. GCs are separated into two anatomic compartments: the dark zone, where B cells divide and undergo somatic hypermutation, and the light zone, where they are selected for affinity-enhancing mutations after interacting with T follicular helper cells. Mayer et al. studied apoptosis reporter mice and found that both GC zones experience very high rates of apoptosis (see the Perspective by Bryant and Hodgkin). However, the underlying mechanisms were distinct and microanatomically segregated. Light-zo ne B cells underwent apoptosis by default unless they were rescued by positive selection. In contrast, apoptotic dark-zone B cells were highly enriched among cells with genes damaged by random antibody-gene mutations. Science, this issue p. eaao2602; see also p. 171 The selection of germinal center B cells by apoptosis is regulated by microanatomically distinct mechanisms. INTRODUCTION Germinal centers (GCs) are transient microanatomic structures that form in lymphoid organs during an immune response. They are the sites of B cell clonal expansion and affinity maturation, a process that leads to the production of high-affinity antibodies. GCs are highly dynamic and contain activated B cells, specialized T follicular helper (TFH) cells, and antigen-trapping follicular dendritic cells. GCs are organized into two functionally distinct compartments: a dark zone (DZ) and a light zone (LZ). The DZ is the site of rapid cell division and random antibody-gene mutation, which is initiated by activation-induced cytidine deaminase (AID). The mutation process leads to the accumulation of a large number of closely related B cells that carry receptors with distinct antigen-binding properties. Once they stop dividing, DZ B cells migrate to the LZ, where their newly generated B cell receptors (BCRs) are tested: GC B cells with relatively higher-affinity receptors capture and process more antigen, leading to positive selection by interaction with TFH cells. The positively selected LZ B cells return to the DZ, where they undergo further cycles of division and mutation. Concomitantly, small numbers of memory B cells and antibody-secreting plasma cells exit the GC. Together, these processes provide the mechanistic basis for affinity maturation, which is essential for effective vaccination and protection from infections. RATIONALE In addition to producing antibody variants, AID expression is also a threat to the genome. AID can produce double-strand breaks that are substrates for chromosome translocations. It can also produce immunoglobulin (Ig) gene missense mutations and deletions or create self-reactive antibodies. These deleterious mutations need to be selected against. Indeed, histologists have long appreciated large numbers of apoptotic nuclei in the specialized tingible body macrophages found in GCs. However, beyond histology, little is known about the exact rate of GC B cell apoptosis and whether it differs in the DZ and LZ of the GC. Moreover, the mechanisms that cause apoptosis, their relative importance in each GC compartment, and their role in GC B cell selection have not been defined. To examine these questions, we created fluorescent apoptosis-indicator mice and used them to enumerate, isolate, and characterize dying cells in the GC. RESULTS We found that apoptosis is prevalent in both the DZ and LZ compartments of GCs throughout the immune response: up to 50% of GC B cells undergo programmed cell death every 6 hours. Single dying GC B cells were isolated, and their antibody genes were cloned, expressed by transient transfection, and tested for antigen binding and other properties. Apoptotic DZ cells were highly enriched for Ig genes damaged by AID, including missense mutations and deletions. By contrast, dying LZ cells primarily expressed intact antibodies with a range of affinities indistinguishable from GC B cells in the live LZ compartment. By experimentally blocking positive selection and by using reporter mice for Myc, a proto-oncogene, as an indicator of positive selection, we found that apoptosis is the default fate for LZ GC B cells that are not actively positively selected. Thus, LZ GC B cells carrying low-affinity BCRs do not preferentially undergo apoptosis. Instead, apoptosis occurs irrespective of BCR-affinity, and LZ B cells carrying high-affinity BCRs are simply more likely to be positively selected. CONCLUSION Apoptosis is a major feature of GC B cell biology and is required to counterbalance the high rate of proliferation and purge B cells that carry deleterious mutations. Although apoptosis occurs in both the DZ and LZ, the underlying mechanisms of apoptosis in each zone are distinct and microanatomically segregated. These insights into GC B biology are relevant for vaccine design, particularly for pathogens that normally evade effective antibody responses. Germinal center B cells expressing an apoptosis indicator. Intravital two-photon microscopy of GC B cells in popliteal lymph nodes of immunized mice (GC B cells, yellow and green; follicular dendritic cell networks, red). The fluorescence resonance energy transfer–based INDIA reporter was used to visualize and purify dying GC B cells. B cells undergo rapid cell division and affinity maturation in anatomically distinct sites in lymphoid organs called germinal centers (GCs). Homeostasis is maintained in part by B cell apoptosis. However, the precise contribution of apoptosis to GC biology and selection is not well defined. We developed apoptosis-indicator mice and used them to visualize, purify, and characterize dying GC B cells. Apoptosis is prevalent in the GC, with up to half of all GC B cells dying every 6 hours. Moreover, programmed cell death is differentially regulated in the light zone and the dark zone: Light-zone B cells die by default if they are not positively selected, whereas dark-zone cells die when their antigen receptors are damaged by activation-induced cytidine deaminase.

中文翻译：

---

生发中心细胞凋亡选择的显微解剖分离

亮区和暗区死亡动力学 生发中心 (GC) 是淋巴器官内的区域，成熟 B 细胞在正常免疫反应期间扩张和分化。GCs 被分成两个解剖隔间：暗区，B 细胞分裂并经历体细胞超突变，和亮区，它们在与 T 滤泡辅助细胞相互作用后被选择用于亲和力增强突变。迈耶等人。研究了细胞凋亡报告小鼠，发现两个 GC 区域都经历了非常高的细胞凋亡率（参见 Bryant 和 Hodgkin 的观点）。然而，潜在的机制是不同的并且在显微解剖学上是分离的。光区 B 细胞默认经历凋亡，除非它们被正选择拯救。相比之下，凋亡暗区 B 细胞在基因被随机抗体基因突变破坏的细胞中高度富集。科学，本期第 3 页。eaao2602; 另见第 171 细胞凋亡对生发中心 B 细胞的选择受显微解剖学不同机制的调节。引言 生发中心 (GC) 是免疫反应期间在淋巴器官中形成的瞬时显微解剖结构。它们是 B 细胞克隆扩增和亲和力成熟的位点，这一过程导致高亲和力抗体的产生。GC 具有高度动态性，包含活化的 B 细胞、特化的 T 滤泡辅助 (TFH) 细胞和捕获抗原的滤泡树突状细胞。GC 分为两个功能不同的隔间：暗区 (DZ) 和亮区 (LZ)。DZ 是快速细胞分裂和随机抗体基因突变的位点，由激活诱导的胞苷脱氨酶 (AID) 启动。突变过程导致大量密切相关的 B 细胞的积累，这些 B 细胞携带具有不同抗原结合特性的受体。一旦停止分裂，DZ B 细胞就会迁移到 LZ，在那里测试它们新生成的 B 细胞受体 (BCR)：具有相对较高亲和力受体的 GC B 细胞捕获和处理更多抗原，通过与 TFH 细胞相互作用导致阳性选择. 阳性选择的 LZ B 细胞返回 DZ，在那里它们经历进一步的分裂和突变循环。同时，少量的记忆 B 细胞和分泌抗体的浆细胞会离开 GC。总之，这些过程为亲和力成熟提供了机制基础，这对于有效接种疫苗和防止感染至关重要。基本原理 除了产生抗体变体，AID 表达也是对基因组的威胁。AID 可以产生作为染色体易位底物的双链断裂。它还可以产生免疫球蛋白 (Ig) 基因错义突变和缺失或产生自反应抗体。需要针对这些有害突变进行选择。事实上，组织学家长期以来一直很欣赏在 GC 中发现的特化可染体巨噬细胞中的大量凋亡细胞核。然而，除了组织学之外，关于 GC B 细胞凋亡的确切率以及它在 GC 的 DZ 和 LZ 中是否存在差异知之甚少。此外，导致细胞凋亡的机制，它们在每个 GC 隔室中的相对重要性，以及它们在 GC B 细胞选择中的作用尚未确定。为了检查这些问题，我们创建了荧光凋亡指示小鼠，并使用它们来枚举、分离和表征 GC 中的垂死细胞。结果我们发现，在整个免疫反应过程中，细胞凋亡在 GC 的 DZ 和 LZ 区室中普遍存在：高达 50% 的 GC B 细胞每 6 小时经历一次程序性细胞死亡。分离单个垂死的 GC B 细胞，克隆其抗体基因，通过瞬时转染表达，并测试抗原结合和其他特性。凋亡的 DZ 细胞高度富集受 AID 破坏的 Ig 基因，包括错义突变和缺失。相比之下，垂死的 LZ 细胞主要表达完整的抗体，其具有与活 LZ 隔室中的 GC B 细胞无法区分的一系列亲和力。通过实验阻断阳性选择并使用报告小鼠 Myc（一种原癌基因）作为阳性选择的指标，我们发现细胞凋亡是未主动阳性选择的 LZ GC B 细胞的默认命运。因此，携带低亲和力 BCR 的 LZ GC B 细胞不会优先发生细胞凋亡。相反，无论 BCR 亲和力如何，都会发生细胞凋亡，携带高亲和力 BCR 的 LZ B 细胞更容易被阳性选择。结论 细胞凋亡是 GC B 细胞生物学的一个主要特征，是平衡高增殖率和清除携带有害突变的 B 细胞所必需的。尽管 DZ 和 LZ 都发生细胞凋亡，但每个区域细胞凋亡的潜在机制是不同的并且在显微解剖学上是分离的。这些对 GC B 生物学的见解与疫苗设计相关，特别是对于通常逃避有效抗体反应的病原体。表达凋亡指示剂的生发中心 B 细胞。免疫小鼠腘窝淋巴结中 GC B 细胞的活体双光子显微镜检查（GC B 细胞，黄色和绿色；滤泡树突状细胞网络，红色）。基于荧光共振能量转移的 INDIA 报告基因用于可视化和纯化垂死的 GC B 细胞。B 细胞在称为生发中心 (GC) 的淋巴器官中解剖学上不同的部位经历快速的细胞分裂和亲和力成熟。体内平衡部分由 B 细胞凋亡维持。然而，细胞凋亡对 GC 生物学和选择的确切贡献尚不明确。我们开发了凋亡指示小鼠，并用它们来可视化、纯化、并表征垂死的 GC B 细胞。细胞凋亡在 GC 中很普遍，每 6 小时就有多达一半的 GC B 细胞死亡。此外，程序性细胞死亡在亮区和暗区受到不同的调节：如果没有积极选择，亮区 B 细胞默认死亡，而暗区细胞在其抗原受体被激活诱导的胞苷脱氨酶损伤时死亡.