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严重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细胞依赖性方面很重要免疫反应，GC动力学的基本方面尚未解决。之后有很长的延迟免疫，通常为5-8天，在同种型开关的抗原特异性B细胞扩展之前卵泡是明显的，组织学不同的GC区域的形成变得可分辨。原因这个延迟尚不清楚。据认为，在T/B边框上与Ag特异性T辅助细胞（Th）互动指示最近激活的抗原特异性B细胞的子集返回卵泡，并立即扩散，形成一个生发中心。此时，免疫反应的T/B协作似乎是取决于CD40连接。但是，因为预测的GC B的直接呈洋内扩张细胞通常并不明显，已经出现了一种替代理论，其中返回卵泡的B细胞已经出现内部在等待卵泡辅助T细胞（TFH）的到来，在大幅更高的时间点上增殖。在这里，我们建议研究B细胞与T辅助细胞（Th）子集的B细胞接触的时间和位置定义导致GC转录程序启动或促进的细胞因子分泌曲线在成熟的GC中发现的独特的区域隔离。 AIM1。将遵循特定特异性B细胞其最初的散热内增殖，同种型开关和GC-表达的精确时机和位置相关的转录因子。我们将确定B中GC转录程序的发作是否细胞与1）TH到达卵泡内部的到来是一致的，2）与相邻的，特定的Th相互作用 T/B边界或3）在这两个位置都改变了IL分泌模式。定义哪些细胞因子是在本地分泌，因此与促进GC的促进，T/B边界的载体特异性T细胞和在免疫后，将在多个时间点评估每个GC子域的表达各种各样的细胞因子。 AIM 2。将成像与载体特异性T细胞接触的特异性B细胞接触随着时间的流逝，插入式插入的多光子显微镜及其运动后的运动图像跟踪分析。我们将可视化这些细胞接触以及它们在不同的迁徙命运 GC开发的阶段以及AIM 1的结果所建议的不同关键位置。AIM3。 CD40/CD40L结合将在体内抑制，以建立Reliant的B细胞接触的功能后果在这种分子相互作用上。 GC B细胞的运动将通过时间分辨出浸润性跟踪抑制CD40/CD40L结合以评估其在建立中的作用后，多光子显微镜显微镜检查维持GC动力学的迁移模式。提出的实验将是向前迈出的重要一步生发中心开发以及疫苗设计的未来研究。