S-Geranylgeranyl-L-glutathione is a ligand for human B cell-confinement receptor P2RY8.

Germinal centres are important sites for antibody diversification and affinity maturation, and are also a common origin of B cell malignancies. Despite being made up of motile cells, germinal centres are tightly confined within B cell follicles. The cues that promote this confinement are incompletely understood. P2RY8 is a Gα13-coupled receptor that mediates the inhibition of migration and regulates the growth of B cells in lymphoid tissues1,2. P2RY8 is frequently mutated in germinal-centre B cell-like diffuse large B cell lymphoma (GCB-DLBCL) and Burkitt lymphoma1,3-6, and the ligand for this receptor has not yet been identified. Here we perform a search for P2RY8 ligands and find P2RY8 bioactivity in bile and in culture supernatants of several mouse and human cell lines. Using a seven-step biochemical fractionation procedure and a drop-out mass spectrometry approach, we show that a previously undescribed biomolecule, S-geranylgeranyl-L-glutathione (GGG), is a potent P2RY8 ligand that is detectable in lymphoid tissues at the nanomolar level. GGG inhibited the chemokine-mediated migration of human germinal-centre B cells and T follicular helper cells, and antagonized the induction of phosphorylated AKT in germinal-centre B cells. We also found that the enzyme gamma-glutamyltransferase-5 (GGT5), which was highly expressed by follicular dendritic cells, metabolized GGG to a form that did not activate the receptor. Overexpression of GGT5 disrupted the ability of P2RY8 to promote B cell confinement to germinal centres, which indicates that GGT5 establishes a GGG gradient in lymphoid tissues. This work defines GGG as an intercellular signalling molecule that is involved in organizing and controlling germinal-centre responses. As the P2RY8 locus is modified in several other types of cancer in addition to GCB-DLBCL and Burkitt lymphoma, we speculate that GGG might have organizing and growth-regulatory roles in multiple human tissues

Cannabinoid receptor 2 positions and retains marginal zone B cells within the splenic marginal zone

In addition to other receptors, including sphingosine-1-phosphate receptor 1, cannabinoid receptor 2 positions mouse marginal zone B cells within the marginal zone and also prevents their loss to the blood

Loss of signalling via Gα13 in germinal center B-cell-derived lymphoma

Germinal centre B-cell-like diffuse large B-cell lymphoma (GCB-DLBCL) is a common malignancy, yet the signalling pathways that are deregulated and the factors leading to its systemic dissemination are poorly defined1,2. Work in mice showed that sphingosine-1-phosphate receptor-2 (S1PR2), a Gα12 and Gα13 coupled receptor, promotes growth regulation and local confinement of germinal centre B cells3,4. Recent deep sequencing studies of GCB-DLBCL have revealed mutations in many genes in this cancer, including in GNA13 (encoding Gα13) and S1PR2 (refs 5,6, 7). Here we show, using in vitro and in vivo assays, that GCB-DLBCL-associated mutations occurring in S1PR2 frequently disrupt the receptor's Akt and migration inhibitory functions. Gα13-deficient mouse germinal centre B cells and human GCB-DLBCL cells were unable to suppress pAkt and migration in response to S1P, and Gα13-deficient mice developed germinal centre B-cell-derived lymphoma. Germinal centre B cells, unlike most lymphocytes, are tightly confined in lymphoid organs and do not recirculate. Remarkably, deficiency in Gα13, but not S1PR2, led to germinal centre B-cell dissemination into lymph and blood. GCB-DLBCL cell lines frequently carried mutations in the Gα13 effector ARHGEF1, and Arhgef1 deficiency also led to germinal centre B-cell dissemination. The incomplete phenocopy of Gα13- and S1PR2 deficiency led us to discover that P2RY8, an orphan receptor that is mutated in GCB-DLBCL and another germinal centre B-cell-derived malignancy, Burkitt's lymphoma, also represses germinal centre B-cell growth and promotes confinement via Gα13. These findings identify a Gα13-dependent pathway that exerts dual actions in suppressing growth and blocking dissemination of germinal centre B cells that is frequently disrupted in germinal centre B-cell-derived lymphoma

S-Geranylgeranyl-L-glutathione is a ligand for human B cell-confinement receptor P2RY8.

Germinal centres are important sites for antibody diversification and affinity maturation, and are also a common origin of B cell malignancies. Despite being made up of motile cells, germinal centres are tightly confined within B cell follicles. The cues that promote this confinement are incompletely understood. P2RY8 is a Gα13-coupled receptor that mediates the inhibition of migration and regulates the growth of B cells in lymphoid tissues1,2. P2RY8 is frequently mutated in germinal-centre B cell-like diffuse large B cell lymphoma (GCB-DLBCL) and Burkitt lymphoma1,3-6, and the ligand for this receptor has not yet been identified. Here we perform a search for P2RY8 ligands and find P2RY8 bioactivity in bile and in culture supernatants of several mouse and human cell lines. Using a seven-step biochemical fractionation procedure and a drop-out mass spectrometry approach, we show that a previously undescribed biomolecule, S-geranylgeranyl-L-glutathione (GGG), is a potent P2RY8 ligand that is detectable in lymphoid tissues at the nanomolar level. GGG inhibited the chemokine-mediated migration of human germinal-centre B cells and T follicular helper cells, and antagonized the induction of phosphorylated AKT in germinal-centre B cells. We also found that the enzyme gamma-glutamyltransferase-5 (GGT5), which was highly expressed by follicular dendritic cells, metabolized GGG to a form that did not activate the receptor. Overexpression of GGT5 disrupted the ability of P2RY8 to promote B cell confinement to germinal centres, which indicates that GGT5 establishes a GGG gradient in lymphoid tissues. This work defines GGG as an intercellular signalling molecule that is involved in organizing and controlling germinal-centre responses. As the P2RY8 locus is modified in several other types of cancer in addition to GCB-DLBCL and Burkitt lymphoma, we speculate that GGG might have organizing and growth-regulatory roles in multiple human tissues

Abnormal disulfide-linked oligomerization results in ER retention and altered signaling by TNFR1 mutants in TNFR1-associated periodic fever syndrome (TRAPS)

Tumor necrosis factor (TNF) receptor–associated periodic syndrome (TRAPS) is an autosomal dominant systemic autoinflammatory disease associated with heterozygous mutations in TNF receptor 1 (TNFR1). Here we examined the structural and functional alterations caused by 9 distinct TRAPS-associated TNFR1 mutations in transfected cells and a mouse “knock-in” model of TRAPS. We found that these TNFR1 mutants did not generate soluble versions of the receptor, either through membrane cleavage or in exosomes. Mutant receptors did not bind TNF and failed to function as dominant-negative inhibitors of TNFR1-induced apoptosis. Instead, TRAPS mutant TNFR1 formed abnormal disulfide-linked oligomers that failed to interact with wild-type TNFR1 molecules through the preligand assembly domain (PLAD) that normally governs receptor self-association. TRAPS mutant TNFR1 molecules were retained intracellularly and colocalized with endoplasmic reticulum (ER) markers. The capacity of mutant receptors to spontaneously induce both apoptosis and nuclear factor κB (NF-κB) activity was reduced. In contrast, the R92Q variant of TNFR1 behaved like the wild-type receptor in all of these assays. The inflammatory phenotype of TRAPS may be due to consequences of mutant TNFR1 protein misfolding and ER retention

The G protein–coupled receptor P2RY8 and follicular dendritic cells promote germinal center confinement of B cells, whereas S1PR3 can contribute to their dissemination

The orphan Gα13-coupled receptor P2RY8 is mutated in human germinal center (GC)–derived lymphomas and was recently found to promote B cell association with GCs in a mouse model. Here we establish that P2RY8 promotes clustering of activated B cells within follicles in a follicular dendritic cell (FDC)–dependent manner. Although mice lack a P2RY8 orthologue, we show that mouse GC B cell clustering is also dependent on FDCs acting to support the function of a Gα13-coupled receptor. Mutations in GNA13 and its downstream effector ARHGEF1 are associated with the development of disseminated GC-derived lymphomas. We find that egress of Gna13 mutant GC B cells from lymph nodes in the mouse depends on sphingosine-1-phosphate receptor-3. These findings provide evidence that FDCs promote GC confinement of both human and mouse GC B cells via Gα13-dependent pathways, and they show that dissemination of Gα13-deficient GC B cells additionally requires an egress-promoting receptor