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

描述（由申请方提供）：对病原体和疫苗的有效免疫应答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细胞依赖性免疫应答中的重要性，但GC动力学的基本方面仍未得到解决。免疫后有一个长的延迟，通常为5-8天，在卵泡内同种型转换的抗原特异性B细胞的扩增是明显的，并且组织学上不同的GC区的形成变得可辨别之前。延迟的原因尚不清楚。据认为，在T/B边界与Ag特异性T辅助细胞（Th）的接合指示最近活化的抗原特异性B细胞的亚群返回卵泡并立即增殖，形成生发中心。此时免疫应答中的T/B协作似乎依赖于CD 40连接。然而，由于预测的GC B细胞的立即滤泡内扩增通常不明显，出现了另一种理论，其中已经返回滤泡内部的B细胞等待滤泡辅助T细胞（Tfh）的到来，在相当晚的时间点增殖。在这里，我们建议调查的时间和位置的B细胞接触辅助性T细胞（Th）的子集，并确定细胞因子分泌的档案，导致启动的GC转录程序或促进独特的区域分离中发现的成熟GC。目的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转录程序的启动是否与1）Th到达卵泡内部，2）与T/B边界处的相邻特异性Th相互作用，或3）这些位置中任一个的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，并因此与GC的促进相关，将在免疫后的多个时间点评估T/B边界和每个GC亚结构域处的载体特异性T细胞的多种细胞因子的表达。目的2，半抗原特异性B细胞与携带者特异性T细胞的接触将通过时间分辨的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和随后的迁移命运，他们促进在GC发展的不同阶段，并在目标1的结果所建议的不同的关键位置。目的3，在体内抑制CD 40/CD 40 L结合，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CD 40/CD 40 L结合后，通过时间分辨活体多光子显微镜跟踪GC B细胞的运动，以评估其在建立维持GC动力学的迁移模式中的作用。拟议的实验将是一个重要的一步，为未来的研究，在老年中心的发展以及疫苗的设计。公共卫生相关性：对病原体和疫苗的有效免疫应答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。本申请提出的实验将回答几个基本问题，即如何启动和适当地调节其他淋巴细胞亚群的生发中心。

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.