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: 7728098
• 负责人: ANN M HABERMAN
• 金额: $ 41.38万
• 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/7728098
• 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（由申请人提供）：对病原体和疫苗的有效免疫反应严重取决于生发中心（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细胞依赖性免疫反应中的重要性，但GC动力学的基本方面仍未解决。免疫后（通常为5-8天）在卵泡内的同种型转换特异性B细胞的扩展之前，有很长的延迟，并且在组织学上不同的GC区域的形成变得可见。此延迟的原因尚不清楚。据认为，在T/B边框处与Ag特异性T辅助细胞（Th）的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增殖，形成生发中心。此时，免疫反应中的T/B协作似乎取决于CD40连接。但是，由于通常不明显的是，由于预测的GC B细胞的直接关联性内扩张并不明显，因此出现了替代理论，其中返回到卵泡内部的B细胞在等待卵泡辅助T细胞（TFH）的到来，在后来的时间点上大大增殖。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，并定义导致GC转录程序启动或促进成熟GC中的独特的区分分离的细胞因子分泌曲线。 AIM 1，触发特异性B细胞将被遵循其初始散热器内增殖，同型开关以及GC-相关转录因子的表达的精确时机和位置。我们将确定B细胞中GC转录程序的发作是否与1）TH到达卵泡内部的到来是一致的，2）与在T/B边界处与相邻的，特定的Th相互作用，还是3）改变了这些位置的IL分泌模式。为了定义哪些细胞因子在局部分泌，因此与GC的促进相关，T/B边界处的载体特异性T细胞以及每个GC子域将在免疫后的多个时间点进行评估，以表达其各种细胞因子。 AIM 2，通过分辨出插入的多光子显微镜及其运动后的动作图像分析，将成像触发特异性B细胞与载体特异性T细胞的接触。我们将可视化这些细胞接触以及它们在GC发育中的不同阶段以及AIM 1结果所建议的不同关键位置所促进的随后的迁移命运。AIM3，CD40/CD40L结合将在体内抑制，以建立依赖于这种分子互动的B细胞接触的功能后果。在抑制CD40/CD40L结合后，将通过时间分辨出的浸润性多光显微镜显微镜来跟踪GC B细胞的运动，以评估其在维持GC动力学的迁移模式中的作用。提出的实验将是未来在生发中心发育以及疫苗设计的未来研究的重要一步。公共卫生相关性：对病原体和疫苗的有效免疫反应严重取决于形成生发中心以形成高亲和力记忆B细胞和浆细胞。该申请提出了实验，该实验将回答有关其他淋巴细胞亚群如何开始和适当调节生发中心的几个基本问​​题。