Abstract
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 lymphoma1,3,4,5,6, 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.
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Data availability
The data that support the findings of this study are available from the authors upon reasonable request. Source Data for experiments involving animal models or tonsil specimens are provided with the paper.
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Acknowledgements
We thank E. Bess and P. Turnbaugh for providing access to their HPLC equipment; Z. Zhang and K. Shokat for providing access to their chemistry equipment and quadrupole time-of-flight mass spectrometer; J. Jespersen and J. Baron for helping to coordinate tonsil specimens; L. Staudt for DLBCL cell lines; N. Barclay for the OX56 hybridoma; J. An for assistance with mouse husbandry; and D. Russell, J. McDonald and J. Wu for discussions. J.G.C. is an investigator of the Howard Hughes Medical Institute. E.L. was supported by the UCSF Biomedical Sciences Graduate Program and NSF grant 1144247. This work was supported in part by NIH grant RO1 AI45073.
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Nature thanks Charles Mackay, Michel Nussenzweig and Hai Qi for their contribution to the peer review of this work.
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E.L., J.R.M. and J.G.C. conceptualized the project. E.L. and J.G.C. designed the experiments, interpreted the results and wrote the manuscript. E.L. performed the experiments. F.D.W. chemically synthesized GGG, acquired high-resolution mass spectrometry data and edited the manuscript. Y.X. cloned enzymes and performed qPCR experiments. J.R.M. cloned OX56-tagged receptors and edited the manuscript.
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Extended data figures and tables
Extended Data Fig. 1 Dependence of P2RY8 bioactivity on albumin and the isoprenoid biosynthetic pathway.
a, Serum-free medium containing the indicated amounts of fatty acid-free BSA was placed on HEK293T cells for 16–18 h. The supernatants from these cultures were combined with CXCL12 in migration medium (1:5 dilution) and tested for P2RY8 bioactivity (n = 5). b, P2RY8 ligand bioassay on 50 kDa concentrate (molecules >50 kDa) versus filtrate (molecules <50 kDa) from serum-starved HEK293T supernatant (left) or raw mouse bile (right) (n = 4). c, Diagram of protein precipitation from pig bile using saturated ammonium sulfate (SAS) and methanol extraction of the SAS protein precipitate. Graph shows P2RY8 ligand bioassay of the SAS supernatant and methanol extracts from the protein precipitate, as indicated by arrows (n = 4). d, P2RY8 ligand bioassay of the two layers of a Folch extraction prepared by adding chloroform and water to the methanol extract of the SAS precipitate described in c (n = 5). e, P2RY8 ligand bioassay on supernatants from Hepa 1-6 or HEK293T cells treated with the indicated inhibitors for 16 h (n = 4, P values determined by one-way ANOVA). f, P2RY8 ligand bioassay on supernatants from HEK293T, HeLa or B16 cells treated with 10 µM mevastatin or vehicle (DMSO) for 16 h (n = 4, P values determined by unpaired two-tailed t-test for the indicated comparisons). Data are pooled from three independent experiments (a–f). Graphs depict mean with s.d. and points represent biological replicates.
Extended Data Fig. 2 HPLC fractionation of P2RY8 bioactivity from bile and Q1 mass spectrometry candidate identification.
a, Preparation of a concentrated bile extract from the Folch upper layer described in Extended Data Fig. 1d using acid precipitation, centrifugation and C18 SPE. b, HPLC chromatograms (blue) showing absorbance at 220 nm for each column used for fractionation. Columns were initially tested with a small amount of extract to determine the interval in which bioactivity eluted. The bioactivity graphs (red) that correspond to 1-min fractions are overlaid for the bioactive interval and represent the percentage of P2RY8+ cells that are inhibited in their migration towards CXCL12 in the P2RY8 ligand bioassay. c, Full scan (Q1) mass spectra of purified fractions from the indicated conditions, in negative-ion mode. Zoomed-in spectra of m/z values of 550–600 are shown directly below each Q1 scan. Data are representative of two (a, b) or one (c) independent experiments.
Extended Data Fig. 3 High-resolution mass spectrometry and fragmentation analysis suggest that the bioactive compound is a derivative of glutathione and geranylgeranyl.
a, Left, positive-ion mode LC–MS total-ion chromatogram of purified bile bioactive fraction (red), overlaid with an adjacent non-bioactive fraction (black). Right, high-resolution mass spectrum from time 1.79 of the active fraction. b, MS/MS fragmentation spectra of glutathione in positive-ion mode (top left) and negative-ion mode (top right), compared with MS/MS spectra of purified bile positive-ion 580.3 (bottom left) and negative-ion 578.3 (bottom right). CE, collision energy. c, Positive-ion mode MS/MS/MS fragmentation spectra of the 273.1 ion present in the MS/MS spectra of GG-PP (top) and purified bile ion 580.3 (bottom; zoomed-in spectra from b). d, Positive-ion mode LC–MS total-ion chromatogram (left) and high-resolution mass spectra (right) from time 1.79 of chemically synthesized GGG. e, Negative-ion mode MS/MS spectra of the 578.3 ion from chemically synthesized GGG. Compare to the MS/MS spectra for the 578.3 ion from purified bile in b. Data are representative of two (b, c, e) or one (a, d) independent experiments.
Extended Data Fig. 4 GGG specifically inhibits migration of P2RY8-expressing WEHI-231 cells.
a, Representative flow cytometry plots of migration-inhibition assays performed with 50 ng ml−1 CXCL12 and 100 nM GGG on WEHI-231 cells transduced with empty vector-GFP, P2RY8-GFP, S1PR2-Thy1.1 or GPR4-Thy1.1. b, Transwell migration-inhibition assay using 500 ng ml−1 CXCL13 and the indicated amounts of GGG for WEHI-231 cells transduced with P2RY8-GFP and empty vector-GFP (n = 4). c, Summarized data for WEHI-231 cells transduced with S1PR2-Thy1.1, GPR4-Thy1.1 and empty vector-GFP from assays of the type in a (S1PR2 and GPR4, n = 3; empty vector, n = 4). Data are representative of two independent experiments (a) and pooled from two independent experiments (b, c). Graphs depict mean with s.d. and points represent biological replicates.
Extended Data Fig. 5 P2RY8 expression and distribution in human tonsil.
a, qPCR for expression of P2RY8 in the indicated subsets sorted from human tonsil, relative to PTPRC. (n = 3) b, Immunofluorescence for P2RY8 (green) and CD4 (red) in PFA-fixed human tonsil sections. Inset depicts P2RY8+ and P2RY8high expressing CD4+ cells within the germinal centre and at the germinal-centre border. Scale bars, 50 µm. c, Intracellular flow cytometry using the anti-P2RY8 antibody from b, which binds the C terminus of P2RY8, on empty vector-GFP- or P2RY8-GFP-transduced WEHI-231 (mouse) cells, compared with rabbit isotype control or no primary antibody staining conditions. d, Intracellular flow cytometry for P2RY8 in tonsil IgD+CD38− follicular B cells, IgD−CD38+ germinal-centre B cells, CXCR5−CD4+ T cells or CXCR5+PD-1+ TFH cells. e, Intracellular flow cytometry for P2RY8 in Ly8 cells edited using CRISPR–Cas9 with a control non-targeting guide (red) or a guide targeting P2RY8 (black). f, TIDE analysis of edited Ly8 cells showing editing efficiency around the expected cut site. g, pAKT levels in DOHH2 cells transduced with either GNA13 or empty vector, treated as in Fig. 3a (n = 5). h, pAKT levels in P2RY8-expressing or control WEHI-231 cells, treated as indicated (n = 9). Data are representative of or pooled from three (a), four (b) or two (d) tonsils; and four (h), two (c, e, g) or one (f) experiments. Graphs depict mean with s.d. Points represent biological replicates. P values determined by one-way ANOVA with Bonferroni’s multiple comparisons test (g, h).
Extended Data Fig. 6 Expression of GGT5 by human tonsil FDCs and fragmentation pattern of S-geranylgeranyl-l-Cys-Gly.
a, P2RY8 ligand bioassay on supernatants from HEK293T cells transfected with the indicated enzymes (n = 4 biological replicates). b, Positive-ion mode MS/MS spectra of the m/z 451.3 metabolite from extracts of the type in Fig. 4d, corresponding to S-geranylgeranyl-l-Cys-Gly. c, Immunohistochemistry for GGT5 or CR2 (brown), in serial sections of human tonsil, counterstained with haematoxylin (blue). Ab, antibody. Scale bars, 200 µm. d, Immunofluorescence for GGT5 (green), CR2 (red) and DAPI (blue) in tonsil sections. Serial sections were stained for P2RY8 (green) and DAPI (blue) to visualize the difference between FDC extensions and germinal-centre B cell membranes. The indicated regions in the top panels (scale bars, 100 µm) are enlarged in the bottom panels (scale bars, 25 µm). e, qPCR for GGT5 expression in the indicated tissues and cells from human tonsil, relative to GAPDH. Points within each category represent individual tonsils (whole tonsil, n = 4; tonsil stroma, n = 4; bulk lymphocytes, n = 4; follicular B cells (Fo B), n = 2; germinal-centre B cells (GC B), n = 3; CXCR5−CD4+ T cells, n = 3; CXCR5intCD4+ T cells, n = 2, TFH cells, n = 3). Data are representative of or pooled from two independent experiments (a, b) or representative of four tonsil specimens (c, d). Graphs depict mean with s.d.
Extended Data Fig. 7 GGT5 is expressed by mouse FDCs.
a, Violin plots from a single-cell RNA sequencing dataset18, showing the relative expression levels of Ggt1, Ggt5, Ggt6 and Ggt7 in the indicated stromal cell (SC) subsets. MRC, marginal reticular cell; PvC, perivascular cell; TRC, T zone reticular cell. b, qPCR for expression of Ggt1, Ggt5, Ggt6 and Ggt7 in whole spleen tissue or spleen stroma, relative to Hprt (n = 3 biological replicates). c, d, RNAscope detection of Ggt5 mRNA (red) counterstained with IgD (brown) in the indicated tissues (spleen, peripheral lymph nodes (LN) and Peyer’s patch) in mice eight days after immunization with SRBCs (c) or in lymph nodes from mice treated with LTβR–Fc and TNFR–Fc fusion proteins or control IgG for four days (d). Serial sections are stained for CR1 (blue) and IgD (brown). Scale bars, 100 µm. Each point in b corresponds to a biological replicate. Data are representative of five (c), two (d) or one (a) biological replicates per condition. Graphs depict mean with s.d. The violin plots in a were generated by a webtool (http://scorpio.ucsf.edu/shiny/LNSC/) that does not display the exact minimum, maximum, centre, percentiles or n numbers for each group.
Extended Data Fig. 8 Controls for transduced B cell co-transfer experiments.
a, Immunofluorescence images tracking the positioning of adoptively transferred B cells overexpressing empty vector–GFP (green), and co-transferred with either empty vector–Thy1.1- or GGT5–Thy1.1-overexpressing B cells, in unimmunized (top) or SRBC-immunized (bottom) mice, relative to endogenous B cells (IgD, blue). b, Quantification of images of the type in a and in Fig. 4h, measuring the average distance (in arbitrary units, A.U.) of GFP+ cells from the centre of B cell follicles using IMARIS software. Each point represents a B cell follicle, and three to four similarly sized follicles were chosen randomly from three mice per condition (n = 10 follicles per condition). Graph depicts mean with s.d. P values determined by one-way ANOVA with Bonferroni’s multiple comparisons test. c, Immunofluorescence images tracking positioning of adoptively transferred B cells overexpressing GGT5 or an empty vector control construct from immunized mice of the type in Fig. 4h, by staining for Thy1.1 (red) relative to endogenous B cells (IgD, blue). Scale bars, 100 µm. Data are representative of three (a, c) biological replicates per condition.
Extended Data Fig. 9 FACS gating strategy and purity.
a, Flow cytometry plots showing the gating scheme that was used to sort the indicated cell subsets from human tonsil, along with post-sort purity. b, For each bioassay performed, representative experiments are graphed as percentage of input migration (that is, the percentage of input cells that migrated) for both the transduced and untransduced WEHI-231 subsets indicated. For Fig. 1j, the C18 SPE concentrates exhibited inhibition of overall migration, probably owing to slight toxicity; however, P2RY8+ cells were more selectively inhibited than P2RY8− cells. The baseline migration across experiments differs based on the growth state of the WEHI-231 cells. Graphs depict mean with s.d.
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Lu, E., Wolfreys, F.D., Muppidi, J.R. et al. S-Geranylgeranyl-l-glutathione is a ligand for human B cell-confinement receptor P2RY8. Nature 567, 244–248 (2019). https://doi.org/10.1038/s41586-019-1003-z
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DOI: https://doi.org/10.1038/s41586-019-1003-z
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