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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

基本信息

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
项目类别：
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

项目摘要

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)形成高亲和力的记忆B细胞和浆细胞。尽管GC在T细胞依赖中的重要性免疫反应，GC动力学的基本方面仍然没有解决。之后有很长一段时间的延误免疫，通常是在同型转换抗原特异性B细胞在体内扩增前5-8天毛囊明显，组织学上可见明显的GC区形成。其原因是这一延迟仍不清楚。据认为，T/B交界处与抗原特异性T辅助细胞(Th)的结合指示最近激活的抗原特异性B细胞子集返回卵泡并立即增殖，形成生发中心。T/B协作在这一点上的免疫反应似乎是依赖CD40结扎。然而，因为预测的GC B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，在这种理论中，B细胞已经返回卵泡内部在于等待滤泡辅助T细胞(TFH)的到来，TFH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确定导致GC转录程序启动或促进的细胞因子分泌特征在成熟的GC中发现的独特的带状分离。目的1.半抗原特异性B细胞将被用于它们最初在卵泡内增殖、同型转换和GC-1表达的准确时间和位置相关转录因子。我们将确定GC转录程序在B细胞中的启动细胞符合1)Th到达卵泡内部，2)与邻近的、特定的Th在 T/B交界处，或3)改变了这两个部位的IL分泌模式。要定义哪些细胞因子是在局部分泌，因此与促进GC、T/B交界处的携带者特异性T细胞和将在免疫后的多个时间点对每个GC亚域的表达进行评估种类繁多的细胞因子。目的2.半抗原特异性B细胞与载体特异性T细胞的接触将被成像用时间分辨活体多光子显微镜及其采集后图像跟踪其运动分析。我们将可视化这些细胞接触以及它们在不同时期促进的迁徙命运目标1.目标3的结果所建议的GC发展阶段和不同的关键位置。体内CD40/CD40L结合将被抑制以建立依赖B细胞接触的功能后果在这种分子相互作用上。GC B细胞的运动将被活体内时间分辨跟踪多光子显微镜抑制CD40/CD40L结合后评估其在建立中的作用支持GC动态的迁移模式。拟议中的实验将是向前迈出的重要一步生发中心发展和疫苗设计方面的未来研究。