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

描述（由申请人提供）：对病原体和疫苗的有效免疫应答严重依赖生发中心（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。免疫后有一个很长的延迟，通常是5-8天，在卵泡内同种型转换抗原特异性B细胞的扩增是明显的，组织学上不同的GC区形成变得可辨别的。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）接触，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。此时免疫应答中的T/B协同作用似乎依赖于CD40连接。然而，由于预测的GC B细胞在滤泡内的立即扩张通常并不明显，因此出现了另一种理论，即返回滤泡内部的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增殖。在这里，我们建议研究B细胞与辅助性T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中发现的独特区带分离的细胞因子分泌谱。目的1，将对半抗原特异性B细胞进行跟踪，以确定其初始滤泡内增殖、同型开关和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以下几个因素同时发生：1)Th到达卵泡内部，2)与T/B边界附近的特异性Th相互作用，或3)这些位置的IL分泌模式发生改变。为了确定哪些细胞因子是局部分泌的，从而与GCs的促进相关，将在免疫后的多个时间点评估T/B边界的载体特异性T细胞和每个GC亚域，以评估它们对各种细胞因子的表达。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，并通过采集后的图像分析跟踪它们的运动。我们将可视化这些细胞接触及其在GC发育的不同阶段和Aim 1的结果所提示的不同关键位置所促进的后续迁移命运。目的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，GC B细胞的运动将通过时间分辨活体多光子显微镜进行跟踪，以评估其在建立维持GC动力学的迁移模式中的作用。所提出的实验将是未来研究生发中心发展和疫苗设计的重要一步。公共卫生相关性：对病原体和疫苗的有效免疫应答严重依赖生发中心的形成，以形成高亲和力记忆的B细胞和浆细胞。本应用程序提出的实验将回答有关生发中心如何开始并适当地由其他淋巴细胞亚群调节的几个基本问题。

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.