Molecular Regulation of Germinal Center B Lymphocytes

描述（由申请人提供）：生发中心（GCs）在免疫或感染后在次级淋巴器官中短暂形成，是提供体液免疫的核心。在GC内，抗原特异性B淋巴细胞表达激活诱导胞苷脱氨酶（AID），诱导体细胞超突变（SMH）和类开关重组（CSR）。这些过程分别随机突变免疫球蛋白位点和切换抗体同型。在突变的GC B细胞中，竞争选择随之而来，高亲和力的B细胞经历选择性扩增并分化为记忆细胞或浆细胞。虽然GC已经被研究了很长时间，但控制高亲和力B细胞选择的事件仍然不清楚。GC在解剖学和功能上被细分为两个不同的区域，亮区（LZ）和暗区（DZ）。在LZ中，B细胞结合抗原，作为免疫复合物保留在滤泡树突状细胞（fdc）上，按其亲和力的比例，并将其加工成与主要组织相容性复合物II （pMHCII）结合的肽，呈递给CD4+ T细胞。通过这种方式，基于pMHCII和T细胞帮助选择高亲和力的GC B细胞，但其发生的机制尚不清楚。此外，除了内吞抗原外，B细胞受体（BCR）在GC选择中的直接作用已被提出，但从未被阐明。由于GC中正在进行的同型交换改变了BCR信令的容量，这个问题变得更加复杂。本研究旨在分析pMHCII水平和同型特异性BCR信号转导如何调节GC B细胞选择机制。为了实现第一个目标，将使用抗原递送系统，以一种暂时可控的方式调节GC B细胞亚群上的pMHCII水平。为了实现第二个目标，将产生新的小鼠，其中BCR的同型可以通过cre重组酶进行切换，而不依赖于抗体亲和力和体细胞超突变。这些问题是GC生物学和体液免疫反应的核心，将通过结合小鼠分子遗传学、传统免疫学技术、流式细胞术和经历GC反应的活体动物的多光子活体成像来解决。

DESCRIPTION (provided by applicant): Germinal centers (GCs) form transiently in secondary lymphoid organs following immunization or infection and are central in providing humoral immunity. Within the GC, antigen-specific B lymphocytes express activation-induced cytidine deaminase (AID), inducing somatic hypermutation (SMH) and class switch recombination (CSR). These processes randomly mutate the immunoglobulin locus and switch antibody isotype, respectively. Competitive selection ensues among mutant GC B cells, with high affinity B cells undergoing selective expansion and differentiating into memory or plasma cells. Although the GC has long been studied, the events controlling selection of high affinity B cells remain obscure. The GC is subdivided into two anatomically and functionally distinct regions, the light (LZ) and dark zones (DZ). In the LZ, B cells bind antigen, retained as immune complexes on follicular dendritic cells (FDCs), in proportion to their affinity and process it for presentation to CD4+ T cells as peptide bound to major histocompatibility complex II (pMHCII). In this manner, high affinity GC B cells are selected based on pMHCII and T cell help, but the mechanisms by which this occurs are not known. Furthermore, a direct role for the B cell receptor (BCR) in GC selection, in addition to endocytosing antigen, has been suggested but never elucidated. This issue is further complicated by ongoing isotype switching in the GC, which changes the BCR signaling capacity. This proposal seeks to analyze how pMHCII levels and isotype-specific BCR signal transduction regulate GC B cell selection mechanisms. Toward the first goal, an antigen delivery system will be used that can modulate pMHCII levels on a subset of GC B cells in a temporally controlled manner. Toward the second goal, novel mice will be generated in which the isotype of the BCR can be switched through cre recombinase, independently of antibody affinity and somatic hypermutation. These problems, central to GC biology and the humoral immune response, will be addressed through a combination of mouse molecular genetics, traditional immunology techniques, flow cytometry, and multiphoton intravital imaging of live animals undergoing GC responses.