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: 7888316
• 负责人: ANN M HABERMAN
• 金额: $ 40.96万
• 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/7888316
• 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(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，以形成高亲和力的记忆B细胞和浆细胞。尽管GC在T细胞依赖的免疫反应中很重要，但GC动力学的基本方面仍未解决。免疫后有一段很长的延迟，通常是5-8天，直到滤泡内明显的同型转换抗原特异性B细胞扩张，并形成组织学上不同的GC区。这一延迟的原因尚不清楚。据认为，与T/B交界处的抗原特异性T辅助细胞(Th)结合，指示最近激活的抗原特异性B细胞亚群返回卵泡并立即增殖，形成生发中心。T/B协同在免疫应答中的作用似乎依赖于CD40的连接。然而，由于预测的GC B细胞在卵泡内的即时扩张通常并不明显，因此出现了另一种理论，即已返回卵泡内部的B细胞等待卵泡辅助T细胞(TFH)的到来，并在相当晚的时间点增殖。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，并确定导致GC转录程序启动或促进成熟GC中发现的独特带状分离的细胞因子分泌谱。目的1、研究半抗原特异性B细胞在卵泡内初始增殖、同型转换和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到达卵泡内部，2)在T/B交界处与邻近的特定Th相互作用，或3)在这两个位置改变IL分泌模式是一致的。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，T/B交界处的携带者特异性T细胞和每个GC亚区将在免疫后的多个时间点进行评估，以了解它们是否表达各种细胞因子。目的利用时间分辨活体内多光子显微镜对半抗原特异性B细胞与载体特异性T细胞的接触进行成像，并通过采集后的图像分析跟踪其运动。我们将可视化这些细胞接触和它们在GC发育的不同阶段和目标1结果所建议的不同关键位置促进的后续迁移命运。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细胞接触的功能后果。在抑制CD40/CD40L结合后，将通过时间分辨活体内多光子显微镜跟踪GC B细胞的移动，以评估其在建立维持GC动力学的迁移模式中的作用。拟议的实验将是未来生发中心开发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，以形成高亲和力的记忆B细胞和浆细胞。这项申请提出的实验将回答几个基本问题，即生发中心是如何开始的，并被其他淋巴细胞亚群适当地调节。