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: 7728098
• 负责人: ANN M HABERMAN
• 金额: $ 41.38万
• 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/7728098
• 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; 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; Time; Vaccine Design; Vaccines; cell motility; cytokine; in vivo; migration; pathogen; programs; public health relevance; research study; segregation; theories; transcription factor

DESCRIPTION (provided by applicant): Effective immune responses to pathogens and vaccines critically depend 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 multiphoton 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. PUBLIC HEALTH RELEVANCE: Effective immune responses to pathogens and vaccines critically depend on the formation of germinal centers to form high affinity memory B cells and plasma cells. This application proposes experiments that will answer several fundamental questions about how germinal centers are begun and appropriately regulated by other lymphocyte subsets.

描述（由申请人提供）：对病原体和疫苗的有效免疫反应关键取决于生发中心（GC）的形成，以形成高亲和力记忆B细胞和浆细胞。尽管 GC 在 T 细胞依赖性免疫反应中很重要，但 GC 动力学的基本方面仍未得到解决。免疫后有很长一段延迟，通常是 5-8 天，然后滤泡内同种型转换的抗原特异性 B 细胞的扩张变得明显，并且组织学上不同的 GC 区的形成变得可辨别。延迟的原因尚不清楚。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 协作似乎依赖于 CD40 连接。然而，由于预测的 GC B 细胞在滤泡内立即扩张通常并不明显，因此出现了另一种理论，其中返回滤泡内部的 B 细胞等待滤泡辅助 T 细胞 (Tfh) 的到来，并在相当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，并定义导致 GC 转录程序启动或促进成熟 GC 中独特的区域分离的细胞因子分泌谱。目标 1，将跟踪半抗原特异性 B 细胞的初始滤泡内增殖、同种型转换和 GC 相关转录因子表达的精确时间和位置。我们将确定 B 细胞中 GC 转录程序的开始是否与 1) Th 到达滤泡内部，2) 与 T/B 边界相邻的特定 Th 相互作用，或 3) 这些位置中任一位置的 IL 分泌模式改变一致。为了确定哪些细胞因子是局部分泌的，从而与 GC 的促进相关联，将在免疫后的多个时间点评估 T/B 边界和每个 GC 子域的载体特异性 T 细胞的多种细胞因子的表达。目标 2，半抗原特异性 B 细胞与载体特异性 T 细胞的接触将通过时间分辨活体多光子显微镜成像，并通过采集后图像分析跟踪它们的运动。我们将可视化这些细胞接触以及它们在GC发育的不同阶段和目标1的结果所暗示的不同关键位置促进的随后的迁移命运。目标3，CD40/CD40L结合将在体内被抑制，以建立依赖于这种分子相互作用的B细胞接触的功能结果。在抑制 CD40/CD40L 结合后，将通过时间分辨活体多光子显微镜追踪 GC B 细胞的运动，以评估其在建立维持 GC 动态的迁移模式中的作用。拟议的实验将是未来生发中心发育和疫苗设计研究的重要一步。公共卫生相关性：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，以形成高亲和力记忆 B 细胞和浆细胞。该申请提出的实验将回答有关生发中心如何开始以及如何受到其他淋巴细胞亚群适当调节的几个基本问​​题。