Molecular Regulation of Germinal Center B Lymphocytes

描述（由申请方提供）：免疫或感染后，在次级淋巴器官中短暂形成生发中心（GC），并在提供体液免疫中发挥重要作用。在GC内，抗原特异性B淋巴细胞表达活化诱导的胞苷脱氨酶（AID），诱导体细胞超突变（SMH）和类别转换重组（CSR）。这些过程分别随机突变免疫球蛋白基因座和转换抗体同种型。竞争性选择在突变型GC B细胞中增强，高亲和力B细胞经历选择性扩增并分化成记忆细胞或浆细胞。虽然GC已经研究了很长时间，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细胞与抗原结合，以免疫复合物的形式保留在滤泡树突细胞（FDC）上，与它们的亲和力成比例，并将其加工为与主要组织相容性复合物II（pMHCII）结合的肽，呈递给CD4+ T细胞。以这种方式，基于pMHCII和T细胞帮助选择高亲和力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，但从未阐明。GC中正在进行的同种型转换使这个问题进一步复杂化，这改变了BCR信号传导能力。本研究旨在分析pMHCII水平和同种型特异性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，将使用一种抗原递送系统，其可以以时间控制的方式调节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的同种型可以通过cre重组酶转换，而不依赖于抗体亲和力和体细胞超突变。这些问题，中央GC生物学和体液免疫反应，将通过小鼠分子遗传学，传统的免疫学技术，流式细胞术和多光子活体成像的活动物经历GC反应的组合来解决。

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.