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 ?-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. !

Public Health Relevance

Germinal center (GC) B cell derived lymphomas such as Burkitt's lymphoma and GCB-DLBCL are aggressive malignancies that are associated with significant morbidity and mortality. Two Ga13-coupled GPCRs, P2RY8 and S1PR2, promote the anatomic confinement and clustering of human GC B cells and together with their effector protein Ga13, have been shown to be mutated in up to 60% of some GC B cell derived lymphomas. We aim to define the mechanism by which the molecule Ggg, a newly discovered ligand for P2RY8, controls germinal center confinement and prevents the development of lymphoma.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Individual Predoctoral NRSA for M.D./Ph.D. Fellowships (ADAMHA) (F30)
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Special Emphasis Panel (ZRG1)
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Gondre-Lewis, Timothy A
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University of California San Francisco
Schools of Medicine
San Francisco
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