Guiding immune cells to the center Germinal centers (GCs) are the site of antibody affinity maturation. The GC response fundamentally depends on contact-dependent signal exchange between antigen-specific T and B lymphocytes. Lu et al. uncovered a repulsive guidance system that inhibits GC recruitment and retention of T follicular helper (TFH) cells while simultaneously promoting their helper activities locally (see the Perspective by Moschovakis and Forster). This system comprises the GC-specific transmembrane ephrin B1 (EFNB1) molecule and two EFNB1 receptors—EPHB4 and EPHB6—expressed by activated T cells, including TFH cells. In the absence of EFNB1 on GC B cells or when EPHB6 was suppressed on T cells, inappropriately large numbers of T cells were recruited to and retained in the GC as a result of relaxed repulsion of TFH cells. Science, this issue p. eaai9264; see also p. 703 A repulsive guidance system is involved in immunological T cell recruitment to the sites of antibody affinity maturation. INTRODUCTION Development of long-term antibody-mediated immunity relies on the germinal center (GC) reaction, in which CD4+ T cells help B cells to proliferate and differentiate into long-lived antibody-producing plasma cells or memory B cells. The GC reaction is orchestrated in a specialized microenvironment and critically depends on guided migratory dynamics of T and B cells. Follicular T helper (TFH) cells, a subset of effector CD4+ helper T cells specialized in supporting the GC reaction, deliver contact-dependent help signals, such as the CD40 ligand, and cytokines, such as interleukin-21 (IL-21), to promote GC B cell proliferation and differentiation. However, exaggerated TFH accumulation and function can cause abnormal GC responses and potentiate autoantibody-mediated inflammatory diseases or even lymphomas. Understanding of how TFH cells are recruited, retained, and functionally regulated in the GC microenvironment is needed for mechanistic insights into long-lived antibody-mediated immunity. Such understanding will inform better design of antibody-based vaccines for microbial infections and therapies for autoimmune diseases. RATIONALE Local mechanisms that regulate TFH cell dynamics and helper functions in GCs are not well defined. Multiple soluble guidance cues sensed by heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors promote TFH confinement toward the center of the B cell follicle of the secondary lymphoid organ, where the GC is typically formed. Contact-dependent guidance mechanisms are also thought to help regulate recruitment and retention of TFH cells in GCs. To search for such mechanisms, we examined the erythropoietin-producing hepatocellular (EPH) receptors and EPH-interacting protein (ephrin, EFN) family members. These molecules are known to guide cell migration during tissue morphogenesis in a contact-dependent manner. RESULTS We found that ephrin-B1 (EFNB1), a class B ephrin, was highly expressed by GC B cells and demarcated the GC tissue domain boundary. Although EFNB1 was not required for initial GC formation or development of TFH cells, its ablation from B cells led to exaggerated local accumulation of TFH cells inside the GC. Dynamic imaging by intravital microscopy revealed that EFNB1 repulsively inhibited GC recruitment and retention of TFH cells, likely as a result of EFNB1-mediated suppression of antigen-specific GC B cell–TFH adhesion. This contact-dependent repulsion required forward signaling through TFH-expressed EPHB6 but not EPHB4 receptor. Unexpectedly, despite the exaggerated GC TFH accumulation, the magnitude of GC responses was largely unchanged, whereas development of plasma cells and affinities of antigen-specific antibodies were actually reduced. By contrast, in a GC mixed with EFNB1-sufficient and -deficient B cells, the latter cells had a competitive advantage in contributing to the plasma cell compartment. These apparently contradicting phenotypes revealed that, by EPHB4-dependent forward signaling, EFNB1 also promotes GC TFH production of IL-21, cytokine that is needed to drive plasma cell formation. Consequently, at the tissue level, EFNB1-deficient GCs lack sufficient IL-21 to support normal plasma cell formation and affinity maturation; in a competitive GC reaction where the same cohort of TFH cells were shared by EFNB1-deficient and -sufficient GC B cells, the role of EFNB1 in suppressing antigen-specific interactions between individual TFH and GC B cells dominates, affording the EFNB1-deficient GC B cells with more contact-dependent help and a competitive advantage. CONCLUSION By revealing a dual role for the GC-expressed EFNB1 molecule, our results uncover a regulatory system that controls GC TFH dynamics and function in the local tissue microenvironment. EFNB1 suppresses T-B adhesion and repulsively inhibits dynamic TFH recruitment and retention in the GC; yet, it also positively promotes IL-21 production by TFH cells as they dwell in the GC at any moment. This counterintuitive combination of negative regulation of residence but positive promotion of effector functions controlled by the same EFNB1 molecule likely helps to ensure that the potentially dangerous GC reaction is productive yet self-limiting. That helper T cell cytokine production is critically modulated in a tissue microenvironment–specific manner by dedicated receptor-ligand systems, the EFNB1-EPHB4 pair in the case of GCs, suggests strategies and targets of manipulation for the development of improved vaccines or therapies. EFNB1 helps germinal center B cells to control TFH cell trafficking and function. EFNB1 repulsively inhibits GC retention of TFH cells while stimulating these cells to produce helper cytokine IL-21 in GCs. In the absence of EFNB1 (left), TFH cells overstay and abnormally accumulate in the GC while their production of IL-21 is reduced, leading to reduced plasma cell formation. The green tracks represent cells that successfully leave the GCs. Follicular T helper (TFH) cells orchestrate the germinal center (GC) reaction locally. Local mechanisms regulating their dynamics and helper functions are not well defined. Here we found that GC-expressed ephrin B1 (EFNB1) repulsively inhibited T cell to B cell adhesion and GC TFH retention by signaling through TFH-expressed EPHB6 receptor. At the same time, EFNB1 promoted interleukin-21 production from GC TFH cells by signaling predominantly through EPHB4. Consequently, EFNB1-null GCs were associated with defective production of plasma cells despite harboring excessive TFH cells. In a competitive GC reaction, EFNB1-deficient B cells more efficiently interacted with TFH cells and produced more bone-marrow plasma cells, likely as a result of gaining more contact-dependent help. Our results reveal a contact-dependent repulsive guidance system that controls GC TFH dynamics and effector functions locally.

中文翻译：

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Ephrin B1 介导的排斥和信号控制生发中心 T 细胞的领土和功能

将免疫细胞引导至中心 生发中心 (GC) 是抗体亲和力成熟的场所。GC 反应从根本上取决于抗原特异性 T 和 B 淋巴细胞之间的接触依赖性信号交换。卢等人。发现了一种排斥性引导系统，该系统抑制 GC 募集和 T 滤泡辅助 (TFH) 细胞的保留，同时在本地促进它们的辅助活动（参见 Moschovakis 和 Forster 的观点）。该系统包含 GC 特异性跨膜 ephrin B1 (EFNB1) 分子和两种 EFNB1 受体——EPHB4 和 EPHB6——由活化的 T 细胞（包括 TFH 细胞）表达。在 GC B 细胞上不存在 EFNB1 或当 T 细胞上的 EPHB6 被抑制时，由于 TFH 细胞的放松排斥，不恰当地大量 T 细胞被募集并保留在 GC 中。科学，这个问题 eaai9264; 另见第。703 排斥引导系统参与免疫 T 细胞向抗体亲和力成熟位点的募集。引言 长期抗体介导免疫的发展依赖于生发中心 (GC) 反应，其中 CD4+ T 细胞帮助 B 细胞增殖并分化为长寿命的产生抗体的浆细胞或记忆 B 细胞。GC 反应是在专门的微环境中精心策划的，并且严重依赖于 T 和 B 细胞的引导迁移动力学。滤泡 T 辅助 (TFH) 细胞是专门支持 GC 反应的效应 CD4+ 辅助 T 细胞的一个子集，提供依赖于接触的帮助信号，例如 CD40 配体和细胞因子，例如白细胞介素 21 (IL-21)，促进GC B细胞增殖和分化。然而，夸大的 TFH 积累和功能会导致异常的 GC 反应并加剧自身抗体介导的炎症性疾病甚至淋巴瘤。需要了解 TFH 细胞如何在 GC 微环境中被募集、保留和功能调节，以便深入了解长寿命抗体介导的免疫。这种理解将为更好地设计基于抗体的微生物感染疫苗和治疗自身免疫性疾病提供信息。基本原理 调节 GC 中 TFH 细胞动力学和辅助功能的局部机制尚未明确定义。异源三聚鸟嘌呤核苷酸结合蛋白 (G 蛋白) 偶联受体感知的多种可溶性指导线索促进 TFH 限制在次级淋巴器官的 B 细胞滤泡中心，GC 通常在此处形成。接触依赖性指导机制也被认为有助于调节 GC 中 TFH 细胞的募集和保留。为了寻找这种机制，我们检查了产生促红细胞生成素的肝细胞 (EPH) 受体和 EPH 相互作用蛋白（ephrin，EFN）家族成员。已知这些分子在组织形态发生过程中以接触依赖性方式引导细胞迁移。结果 我们发现 ephrin-B1 (EFNB1) 是一种 B 类 ephrin，在 GC B 细胞中高度表达，并划定了 GC 组织域边界。尽管 EFNB1 不是 TFH 细胞初始 GC 形成或发育所必需的，但它从 B 细胞中消融导致 GC 内 TFH 细胞的局部积累过度。活体显微镜动态成像显示 EFNB1 排​​斥抑制 GC 募集和 TFH 细胞的保留，可能是 EFNB1 介导的抗原特异性 GC B 细胞-TFH 粘附抑制的结果。这种依赖于接触的排斥需要通过 TFH 表达的 EPHB6 而不是 EPHB4 受体的正向信号传递。出乎意料的是，尽管 GC TFH 积累过多，但 GC 反应的程度基本没有变化，而浆细胞的发展和抗原特异性抗体的亲和力实际上降低了。相比之下，在与 EFNB1 充足和缺乏的 B 细胞混合的 GC 中，后一种细胞在形成浆细胞室方面具有竞争优势。这些明显矛盾的表型表明，通过依赖 EPHB4 的正向信号传导，EFNB1 还促进 GC TFH 产生 IL-21，IL-21 是驱动浆细胞形成所需的细胞因子。因此，在组织水平上，EFNB1 缺陷型 GC 缺乏足够的 IL-21 来支持正常的浆细胞形成和亲和力成熟；在竞争性 GC 反应中，EFNB1 缺陷和充足的 GC B 细胞共享同一组 TFH 细胞，EFNB1 在抑制个体 TFH 和 GC B 细胞之间抗原特异性相互作用中的作用占主导地位，提供了 EFNB1 缺陷的 GC B 细胞具有更多依赖于接触的帮助和竞争优势。结论 通过揭示 GC 表达的 EFNB1 分子的双重作用，我们的结果揭示了一个调控系统，该系统控制局部组织微环境中的 GC TFH 动力学和功能。EFNB1 抑制 TB 粘附并排斥性抑制 GC 中的动态 TFH 募集和保留；然而，它也积极促进 TFH 细胞产生 IL-21，因为它们随时驻留在 GC 中。这种对滞留的负调节与效应器功能的正向促进的反直觉组合，由相同的 EFNB1 分子控制，可能有助于确保潜在危险的 GC 反应是高效的，但又是自限性的。辅助性 T 细胞细胞因子的产生通过专门的受体-配体系统以组织微环境特异性方式受到严格调节，在 GC 的情况下，EFNB1-EPHB4 对为开发改进的疫苗或疗法提供了操作策略和目标。EFNB1 帮助生发中心 B 细胞控制 TFH 细胞的运输和功能。EFNB1 排​​斥抑制 TFH 细胞的 GC 保留，同时刺激这些细胞在 GC 中产生辅助细胞因子 IL-21。在没有 EFNB1（左）的情况下，TFH 细胞在 GC 中过度停留并异常积累，同时它们的 IL-21 产生减少，导致浆细胞形成减少。绿色轨道代表成功离开 GC 的细胞。滤泡 T 辅助 (TFH) 细胞在局部协调生发中心 (GC) 反应。调节其动力学和辅助功能的局部机制尚未明确定义。在这里，我们发现 GC 表达的 ephrin B1 (EFNB1) 通过 TFH 表达的 EPHB6 受体发出信号，排斥性抑制 T 细胞与 B 细胞的粘附和 GC TFH 保留。同时，EFNB1 主要通过 EPHB4 发出信号，促进 GC TFH 细胞产生白细胞介素 21。因此，尽管含有过多的 TFH 细胞，但 EFNB1-null GC 与浆细胞的产生缺陷有关。在竞争性 GC 反应中，EFNB1 缺陷的 B 细胞更有效地与 TFH 细胞相互作用并产生更多的骨髓浆细胞，这可能是因为获得了更多依赖于接触的帮助。我们的结果揭示了一种依赖于接触的排斥引导系统，该系统可在本地控制 GC TFH 动力学和效应器功能。