In vivo imaging of T and B cell interactions in germinal center initiation

生发中心启动过程中 T 细胞和 B 细胞相互作用的体内成像

• 批准号: 8286885
• 负责人: ANN M HABERMAN
• 金额: $ 40.55万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-15 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8286885
• 关键词: Affinity; Antibodies; Antigens; B Cell Proliferation; B-Lymphocytes; Back; Behavior; Binding; CD4 Positive T Lymphocytes; Cell Communication; Cells; Collaborations; Development; Eating; Event; Fab Immunoglobulins; Future; Haptens; Helper-Inducer T-Lymphocyte; Histologic; Image; Image Analysis; Immune response; Immunization; Immunoglobulin Class Switching; In Situ Hybridization; Injection of therapeutic agent; Interleukin-10; Interleukin-4; Lead; Ligation; Light; Location; Lymphocyte Subset; Mediating; Memory B-Lymphocyte; Microscopy; Movement; Mus; Pattern; Plasma Cells; Population; Proliferating; Reaction; Reporter; Role; Site; Staging; Stimulus; Structure of germinal center of lymph node; T-Lymphocyte; TNFRSF5 gene; TNFSF5 gene; Time; Vaccine Design; Vaccines; cell motility; cytokine; in vivo; migration; pathogen; programs; research study; segregation; theories; transcription factor

Effective immune responses to pathogens and vaccines critically depends on the formation of germinal centers (GC) to form high affinity memory B cells and plasma cells. Despite the importance of GCs in T cell dependent immune responses, fundamental aspects of GC dynamics remain unresolved. There is a long delay after immunization, typically 5-8 days, before expansion of isotype-switched antigen-specific B cells within the follicle is evident and the formation of histologically distinct GC zones becomes discernable. The reason for this delay remains unclear. It is thought that engagement with Ag specific T helper cells (Th) at the T/B border instructs a subset of recently activated antigen specific B cells to return to the follicle and immediately proliferate, forming a germinal center. T/B collaboration at this point in the immune response appears to be dependent upon CD40 ligation. However, because the predicted immediate intra-follicular expansion of GC B cells is not typically evident, an alternative theory has emerged in which B cells that have returned to the follicle interior lie in wait for the arrival of follicular helper T cells (Tfh), proliferating at a substantially later timepoint. Here we propose to investigate the timing and location of B cell contacts with T helper cells (Th) subsets and to define the cytokine secretion profiles that lead to the initiation of the GC transcriptional program or promote the unique zonal segregation found in mature GCs. Aim 1. Hapten specific B cells will be followed for the precise timing and location of their initial intra-follicular proliferation, isotype switch, and expression of GC- associated transcription factors. We will determine whether the onset of the GC transcriptional program in B cells is coincident with either 1) the arrival of Th to the follicle interior, 2) interaction with adjacent, specific Th at the T/B border, or 3) altered IL secretion patterns at either of these locations. To define which cytokines are secreted locally, and hence correlate with the promotion of GCs, carrier specific T cells at the T/B border and each of the GC subdomains will be assessed at multiple time points post immunization for their expression of a wide variety of cytokines. Aim 2. Contact of hapten specific B cells with carrier specific T cells will be imaged by time resolved intravital multphoton microscopy and their movement tracked with post-acquisition image analysis. We will visualize these cellular contacts and the subsequent migratory fates they promote at different stages in GC development and at the distinct key locations suggested by the results of Aim 1. Aim 3. CD40/CD40L binding will be inhibited in vivo to establish the functional consequences of B cell contacts reliant on this molecular interaction. The movement of GC B cells will be tracked by time resolved intravital multiphoton microscopy after inhibition of CD40/CD40L binding to assess its role in the establishment of migration patterns that sustain GC dynamics. The proposed experiments will be an important step forward for future studies in germinal center development as well as vaccine design.

对病原体和疫苗的有效免疫反应关键取决于生发中心的形成 (GC)形成高亲和力记忆B细胞和浆细胞。尽管 GC 在 T 细胞依赖性中很重要 免疫反应，GC 动力学的基本方面仍未解决。之后有很长的延迟 免疫接种，通常是 5-8 天，然后在同种型转换的抗原特异性 B 细胞在 滤泡是明显的，并且组织学上不同的 GC 区的形成变得可辨别。原因是 这种延迟仍不清楚。据认为，T/B 边界处与 Ag 特异性 T 辅助细胞 (Th) 的结合 指示最近激活的抗原特异性 B 细胞子集返回滤泡并立即 增殖，形成生发中心。此时免疫反应中的 T/B 合作似乎是 依赖于 CD40 连接。然而，由于预测 GC B 会立即在卵泡内扩张 细胞通常并不明显，另一种理论已经出现，其中 B 细胞已返回滤泡 内部等待滤泡辅助 T 细胞 (Tfh) 的到来，并在较晚的时间点进行增殖。 在这里，我们建议研究 B 细胞与 T 辅助细胞 (Th) 亚群接触的时间和位置，以及 定义导致 GC 转录程序启动或促进的细胞因子分泌谱 成熟GC 中发现的独特的区域偏析。目标 1. 将跟踪半抗原特异性 B 细胞 其初始滤泡内增殖、同种型转换和 GC- 表达的精确时间和位置 相关转录因子。我们将确定GC转录程序是否在B中开始 细胞与 1) Th 到达毛囊内部，2) 与邻近的特定 Th 相互作用一致 T/B 边界，或 3) 改变这些位置的 IL 分泌模式。定义哪些细胞因子 局部分泌，因此与 GC 的促进相关，T/B 边界的载体特异性 T 细胞和 每个 GC 子域将在免疫后的多个时间点评估其表达 细胞因子种类繁多。目标 2. 对半抗原特异性 B 细胞与载体特异性 T 细胞的接触进行成像 通过时间分辨活体多光子显微镜及其通过采集后图像跟踪的运动 分析。我们将可视化这些细胞接触以及它们在不同的地方促进的随后的迁移命运 目标 1 和目标 3 的结果表明 GC 发展的各个阶段以及不同的关键位置。 CD40/CD40L 结合将在体内受到抑制，以建立依赖于 B 细胞接触的功能结果 关于这种分子相互作用。 GC B 细胞的运动将通过时间分辨的活体追踪 抑制 CD40/CD40L 结合后的多光子显微镜评估其在建立 维持 GC 动态的迁移模式。拟议的实验将是向前迈出的重要一步 未来对生发中心发育和疫苗设计的研究。

项目成果

Germinal center B cell development has distinctly regulated stages completed by disengagement from T cell help.

生发中心 B 细胞的发育具有明显的调节阶段，通过脱离 T 细胞的帮助而完成。

• 发表时间: 2017-05-12
• 影响因子: 7.7
• 作者: Zhang, Ting;Gonzalez, David G;Cote, Christine M;Kerfoot, Steven M;Deng, Shaoli;Cheng, Yuqing;Magari, Masaki;Haberman, Ann M
• 通讯作者: Haberman, Ann M

Dynamic expression of BCL6 in murine conventional dendritic cells during in vivo development and activation.

小鼠常规树突状细胞在体内发育和激活过程中 BCL6 的动态表达。

• 发表时间: 2014
• 影响因子: 3.7
• 作者: Zhang, Ting;Liu, Dong;Calabro, Samuele;Eisenbarth, Stephanie C;Cattoretti, Giorgio;Haberman, Ann M
• 通讯作者: Haberman, Ann M