Dissecting the role of geranylgeranyl glutathione in the germinal center response

剖析香叶基香叶基谷胱甘肽在生发中心反应中的作用

• 批准号: 10304180
• 负责人: Antonia Gallman
• 金额: $ 4.52万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-12-14 至 2022-09-13
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10304180
• 关键词: Address; Affinity; Anabolism; Anatomy; Antibody Affinity; Antibody Response; Antigens; B-Lymphocytes; Burkitt Lymphoma; Cell Line; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Coupled; Data; Development; Doctor of Philosophy; Ensure; Enzymes; Event; Evolution; Flow Cytometry; Follicular Dendritic Cells; Fostering; G-Protein-Coupled Receptors; Gamma-glutamyl transferase; Generations; Glutathione; Glutathione Metabolism Pathway; Glutathione S Transferase A; Goals; Growth; Health; Helper-Inducer T-Lymphocyte; Human; Image; Immune; Immune response; Immune system; Immunofluorescence Microscopy; Immunoglobulin Somatic Hypermutation; Island; Knockout Mice; Knowledge; Lead; Ligands; Lymphoid Cell; Lymphoid Tissue; Lymphoma; Malignant Neoplasms; Malignant neoplasm of prostate; Measures; Mediating; Metabolism; Morbidity - disease rate; Mus; Mutate; Mutation; Organ Culture Techniques; Pathway interactions; Pattern; Play; Production; Proteins; Regulation; Role; Signaling Molecule; Site; Slice; Specificity; Structure of germinal center of lymph node; Techniques; Testing; Therapeutic; Time; Tissues; Tonsil; Work; base; cBioPortal; cell type; chemokine; design; experimental study; human migration; in vivo; inhibitor; insight; knock-down; large cell Diffuse non-Hodgkin' s lymphoma; malignant stomach neoplasm; migration; mortality; novel; overexpression; prevent; receptor; response; therapeutic development; two photon microscopy; vaccine development; vaccine immunotherapy

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. !

项目总结/摘要 作为协调高亲和力抗体产生的关键位点，生发中心（GC）在免疫反应中起着重要作用。 在建立有效的体液免疫应答中的重要作用。GC的特征在于络合物， 趋化因子驱动的细胞组织，GC B细胞被严格限制在这个解剖空间，缺乏 再循环的能力。这种限制促进了GC B细胞与辅助性T滤泡细胞的相互作用 细胞（Tfh）和抗原负载的滤泡树突状细胞（FDC），并确保B细胞选择和体细胞 超突变事件发生在良好控制的微环境中。此外，限制被假设为 促进GC作为独立的“细胞岛”的发展，允许不同的高分子的进化， 亲和克隆因此，GC限制被认为允许产生抗体应答，而不是产生抗体应答。 不仅亲和力高，而且特异性多样。我们实验室过去的工作提供了两个Ga 13- 偶联的GPCR，P2 RY 8和S1 PR 2，促进人GC B细胞在细胞内的限制和聚集。 GC通过抑制它们的向外迁移。这些受体及其下游效应子Ga 13可以突变， 在高达60%的GC B细胞来源的淋巴瘤中，强调了它们在人类健康中的重要作用。实验室 最近鉴定了P2 RY 8的内源性配体为香叶基香叶基谷胱甘肽（GGg），显示GGg抑制 人GC B细胞和Tfh细胞的迁移，具有nM效力。然而，酶和转运蛋白 参与这种新的细胞间信号分子的体内产生、输出和降解， 尚未定义。我们的实验室已经发现γ-谷氨酰转移酶-5（Ggt 5）酶能够降解GGG， 证实了初级卵泡和GC内FDCs表达这种酶。初步数据还 支持转运蛋白Abcc 1可能控制Ggg从细胞中的释放。我攻读博士学位的目标 这一建议是为了阐明新发现的代谢产物Ggg在GC反应中的作用。 根据初步数据，我假设，精确控制分布的GGG内 卵泡是限制GC细胞所必需的，因此也是有效的B细胞所必需的 反应在目标1中，我将定义参与GGG代谢的酶和转运蛋白，并检查它们的功能。 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Ggt 5 KO小鼠中的梯度来促进GC限制。在目标3中，我将 使用扁桃体切片器官培养物表征人GC中P2 RY 8-GGg轴的功能。这项工作将 为GGG梯度如何在体内产生提供了新的机制理解，并可能导致 鉴于P2 RY 8在一系列的癌症中的频繁突变， 癌症和广泛的细胞类型产生Ggg的能力，很可能还有其他情况下， ggg介导的限制是重要的。我的研究准备提供新的见解， 免疫系统内外的限制途径。 !

项目成果

P2RY8 variants in lupus patients uncover a role for the receptor in immunological tolerance.

狼疮患者的 P2RY8 变异揭示了受体在免疫耐受中的作用。

• DOI: 10.1084/jem.20211004
• 发表时间: 2022-01-03
• 期刊: The Journal of experimental medicine
• 作者: He Y;Gallman AE;Xie C;Shen Q;Ma J;Wolfreys FD;Sandy M;Arsov T;Wu X;Qin Y;Zhang P;Jiang S;Stanley M;Wu P;Tan J;Ding H;Xue H;Chen W;Xu J;Criswell LA;Nititham J;Adamski M;Kitching AR;Cook MC;Cao L;Shen N;Cyster JG;Vinuesa CG
• 通讯作者: Vinuesa CG