Dissecting the role of geranylgeranyl glutathione in the germinal center response

项目摘要/摘要 作为高亲和力抗体产生的关键部位，生发中心发挥着重要的作用。 在建立有效的体液免疫反应方面发挥重要作用。GC的特征是复杂的， 趋化因子驱动的细胞组织，GC B细胞被紧紧地限制在这个解剖空间，缺乏 循环的能力。这种限制促进了GC B细胞与支持T滤泡辅助细胞的相互作用 细胞(TFH)和抗原负载的滤泡树突状细胞(FDCs)，并确保B细胞选择和体细胞 超突变事件发生在受控良好的微环境中。此外，监禁是假想的 促进GC作为单独的“细胞孤岛”的发展，允许不同的高压演变 亲和克隆。因此，GC限制被认为允许产生不是的抗体反应 不仅亲和力高，而且特异性也不同。我们实验室过去的工作提供了证据，证明两个Ga13- 偶联的GPCRs，P2RY8和S1PR2，促进人GC B细胞在 GC通过抑制其向外迁移。这些受体及其下游效应器Ga13可以突变 在一些GC B细胞来源的淋巴瘤中，高达60%的病例，强调了它们在人类健康中的重要作用。实验室 最近确定了P2RY8的内源性配体为香叶基-谷胱甘肽(GGG)，显示GGG抑制 具有NM活性的人GC B细胞和Tfh细胞的迁移然而，酶和转运蛋白 参与体内这种新的细胞间信号分子的产生、输出和降解 还没有被定义。我们实验室已经发现了γ-谷氨酰转移酶-5(GGT5)能够降解GGG和 FDC在初级卵泡和GC中证实了该酶的表达。初步数据还 支持转运体Abcc1可能控制细胞中GGG的释放。我攻读博士学位的目标 这项建议是为了阐明新发现的代谢物GGG在GC反应中的作用。 根据初步数据，我假设精确控制的GGG在 卵泡是限制GC细胞，从而形成有效的B细胞所必需的 回应。在目标1中，我将定义参与GGG代谢的酶和转运体，并研究它们的 体内分布，以了解GGG梯度是如何建立的。在目标2中，我将确定 GGG梯度通过破坏GGT5KO小鼠的梯度促进GC限制。在《目标3》中，我会 用扁桃体切片器官培养法研究人GCs中P2RY8-GGG轴的功能。这项工作将 为GGG梯度是如何在体内产生并可能导致 设计用来调节它的治疗方法的发展。鉴于P2RY8基因在一系列 癌症和多种细胞类型产生GGG的能力，很可能还有其他背景 GGG介导的禁闭很重要。我的研究准备为这一角色提供新的洞察力 免疫系统内和免疫系统以外的限制途径。 好了！

PROJECT SUMMARY / ABSTRACT As the key site for orchestrating the production of high affinity antibody, the germinal center (GC) plays an important role in mounting effective humoral immune responses. The GC is characterized by a complex, chemokine-driven organization of cells, and GC B cells are tightly confined to this anatomic space, lacking the ability to recirculate. This confinement fosters interactions of the GC B cells with supporting T follicular helper cells (Tfh) and antigen loaded follicular dendritic cells (FDCs), and ensures B cell selection and somatic hypermutation events occur in a well-controlled microenvironment. Furthermore, confinement is hypothesized to contribute to the development of GCs as separate `cellular islands,' allowing the evolution of distinct high affinity clones. Thus, GC confinement is thought to allow for the generation of an antibody response that is not only high in affinity but also diverse in specificity. Past work in our lab provided evidence that two Ga13- coupled GPCRs, P2RY8 and S1PR2, promote the confinement and clustering of human GC B cells within the GC by inhibiting their outward migration. These receptors and their downstream effector Ga13 can be mutated in up to 60% of some GC B cell-derived lymphomas, underscoring their essential role in human health. The lab recently identified the endogenous ligand for P2RY8 as geranylgeranyl-glutathione (Ggg), showing Ggg inhibits migration of human GC B cells and Tfh cells with nM potency. However, the enzymes and transporters involved in the in vivo production, export and degradation of this novel intercellular signaling molecule have not yet been defined. Our lab has found the γ-glutamyltransferase-5 (Ggt5) enzyme capable of degrading Ggg and confirmed the expression of this enzyme by FDCs within primary follicles and GCs. Preliminary data also supports that the transporter Abcc1 may be controlling release of Ggg from cells. The goal of my PhD studies and this proposal is to elucidate the role of the newly discovered metabolite Ggg in the GC response. Based on preliminary data, I hypothesize that the precisely controlled distribution of Ggg within the follicle is necessary for the confinement of GC cells and thus for the mounting of an effective B cell response. In Aim 1, I will define the enzymes and transporters involved in Ggg metabolism and examine their distribution in vivo to understand how the Ggg gradient is established. In Aim 2, I will determine the role of the Ggg gradient in promoting GC confinement via disruption of the gradient in Ggt5 KO mice. In Aim 3, I will characterize the function of the P2RY8-Ggg axis in human GCs using tonsil slice organ culture. This work will provide new mechanistic understanding for how the Ggg gradient is generated in vivo and may lead to the development of therapeutics designed to modulate it. Given the frequent mutation of P2RY8 in a spectrum of cancers and the ability of a wide range of cell types to produce Ggg, it is likely there are other contexts in which Ggg-mediated confinement is important. My studies are poised to provide new insight into the role of this confinement pathway in the immune system and beyond. !