Abstract
Plasma cells produce large quantities of antibodies and so play essential roles in immune protection1. Plasma cells, including a long-lived subset, reside in the bone marrow where they depend on poorly defined microenvironment-linked survival signals1. We show that bone marrow plasma cells use the ligand-gated purinergic ion channel P2RX4 to sense extracellular ATP released by bone marrow osteoblasts through the gap-junction protein pannexin 3 (PANX3). Mutation of Panx3 or P2rx4 each caused decreased serum antibodies and selective loss of bone marrow plasma cells. Compared to their wild-type counterparts, PANX3-null osteoblasts secreted less extracellular ATP and failed to support plasma cells in vitro. The P2RX4-specific inhibitor 5-BDBD abrogated the impact of extracellular ATP on bone marrow plasma cells in vitro, depleted bone marrow plasma cells in vivo and reduced pre-induced antigen-specific serum antibody titre with little posttreatment rebound. P2RX4 blockade also reduced autoantibody titre and kidney disease in two mouse models of humoral autoimmunity. P2RX4 promotes plasma cell survival by regulating endoplasmic reticulum homeostasis, as short-term P2RX4 blockade caused accumulation of endoplasmic reticulum stress-associated regulatory proteins including ATF4 and B-lineage mutation of the pro-apoptotic ATF4 target Chop prevented bone marrow plasma cell demise on P2RX4 inhibition. Thus, generating mature protective and pathogenic plasma cells requires P2RX4 signalling controlled by PANX3-regulated extracellular ATP release from bone marrow niche cells.
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Full scans for gels are provided in Supplementary Fig. 1. Source data are provided with this paper.
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Acknowledgements
We thank M. Cancro, A. Nussenzweig, S. John and V. Smith-Shapiro for reviewing this manuscript, D. Hildeman and A. Marshak-Rothstein for helpful discussions and M. Shlomchik for providing CD20-TAM-Cre mice. We also thank M. Cam and D. Fonseca for help with statistics, H. Averman for help with histological analysis and the UPenn Flow Cytometry and Cell Sorting facility. This work was supported by NIH grant nos. R21 AI161931, RO1 AI139123 and RO1 AI175185 and an ASPIRE award from the Mark Foundation for Cancer Research to D.A., KAKENHI of Japan grant nos. 16KK0196 and 19K22698 to M.I., NIH grant nos. T32-HL07439 and T32AI007632 to Z.H. and J.L., respectively, an Amyloidosis Foundation Research Grant to Z.H. and the Intramural Research Program of the Center for Cancer Research at the NCI (A.B.).
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M.I., D.A. and A.B. designed the experiments. M.I., Z.H., Y.Z., A.D., C.R. and J.L. performed the experiments including all ELISPOT, ELISA and flow cytometry studies. M.L. performed bioinformatic analyses. M.I., D.A. and A.B. wrote the manuscript with input from all other co-authors.
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Extended data figures and tables
Extended Data Fig. 1 Panx3 mutation does not perturb developing or mature myeloid and lymphoid cell populations.
(a) Numbers of viable BM cells in 16-week-old Panx3+/+ and Panx3−/− mice. (b–e) Evaluation of the indicated BM myeloid and B-lineage cells (b), thymocytes (c), T and B cells in spleen (d) and peritoneal B1 cells (e). (b) Numbers of macrophages (CD115− Gr1− F4/80+), Gr1+ and Gr1− monocytes (CD115+ Gr1high, CD115− Gr1low F4/80+/− respectively), neutrophils (CD115− Gr1high), pro- & pre-B cells (CD19+ IgM− CD43+/−) and immature (CD19+ IgM+ IgD−) and mature (CD19+ IgM+ IgD+) B cells. (c-e) Numbers of CD4 and CD8 single positive thymocytes (c), splenic CD4 and CD8 T cells and mature (CD19+ IgD+) B cells (d) and peritoneal cavity CD19 + CD43 + B1 B cells (e) in 5-month-old Panx3+/+ and Panx3−/− mice. (See Extended Figs. 2 and 3 for flow cytometry gating strategies for BM and spleen cells). (n = 6/grp for panels a-d, n = 3/grp for panel e). (f, g) CD138+ Sca-1+ PCs in the BM (f) and spleen (g) in 16-week-old Panx3+/+ and Panx3−/− mice (n = 6/grp). Plots in (f) show additional analysis of newly formed (B220+) and mature (B220−) PCs. All bar graphs are means. For panels (a,b), *, p = 0.0022. All p values were derived from two-tailed Mann–Whitney tests, without any adjustment for multiple comparisons. The experiments in panels (a-d) and (f-g) were performed at least thrice and the experiments in panel (e) were performed twice.
Extended Data Fig. 4 Panx3 and eATP support BM PCs.
(a) Schematic for experiments wherein fresh BM cells were cultured with pre-induced osteoblastic cells for 4 days before ELISPOT analyses. (b) Quantitative RT-PCR for CXCL12, SCF, IL6 and APRIL expression with osteoblastic calvarial cells from Panx3+/+ and Panx3−/− mice after 14 days culture as in (a). Graph shows means for technical replicates; n = 3. A control Panx3+/+ sample was set to 1 for each set of measurements and the other measurements were recalculated relative to that sample. All p values were derived from two-tailed Mann–Whitney tests, without any adjustment for multiple comparisons. The experiments in panel b were performed twice.
Extended Data Fig. 5 P2rX inhibitors deplete BM PCs.
(a) Representative flow cytometry data for BM PCs in WT mice given DMSO alone (Control) or Suramin (2.0 mg/kg), PPADS (2.5 mg/kg), or both Suramin and PPADS thrice over seven days before analysis. Representative of 3 mice per group and three separate experiments. (b) Expression of P2rX and P2rYfamily members in BM PCs (ImmGen data, see Methods). (c) Annotated UMAP visualizing naïve and memory B cells and plasma cells identified in adult human bone marrow (left) with Heatmap visualizing averaged and gene-wise scaled gene expression for each population (right) (see Methods).
Extended Data Fig. 6 Selective impact of B-lineage P2rX4 mutation on BM PCs.
(a) Flow cytometric analyses for BM B220+/− CD138high PCs and numbers of splenic PCs (Dump- IgD- CD138+ Sca-1+) (b, c) Cell numbers for plasma cells in spleen (b) or indicated cell populations in spleen and BM (c) and peritoneal cavity CD19+ CD43+ B1 B cells (d) in 5-month-old WT;Mb1-Cre and P2rX4f/f;Mb1-Cre adults. (For panels (a-c) n = 6/group and for panel (d) n = 3/group). (e,f) Analysis of B220+/− CD138high BM PCs (e) and all splenic PCs (CD138high Sca-1+) (f) in WT;CD20-TAM-Cre and P2rX4f/f; CD20-TAM-Cre adults. (g) Numbers of the indicated T and B cell subsets in the spleen and developing B cells in BM of the indicated mice. (h) ELISPOT analyses of IgG-secreting BM cells from CD20-TAM-Cre and P2rX4f/f;CD20-TAM-Cre mice fed tamoxifen-laced chow for 4 weeks previously. BM cells from tamoxifen-fed mice were cultured for 2 days with or without adding 100μM ATP before addition to ELISPOT assay. For panels (e-h) n = 6/grp. All bar graphs are means. For panels (a-h) experiments were performed at least thrice. *, p = 0.0022. All p values were derived from two-tailed Mann–Whitney tests, without any adjustment for multiple comparisons.
Extended Data Fig. 7 Induced B-lineage-restricted P2rX4 mutation in mature BM PCs.
(a) Cre expression in B220− BM LLPCs of CD20-TAM-Cre adults. Data are representative plots of n = 3 mice/grp. (b) P2rX4 genomic PCR for sorted T cells, B cells and B220− PCs of CD20-TAM-Cre adult mice fed tamoxifen for 1 week (n = 3). HIF1α used as a DNA loading control. BM B220− PCs were sorted sequentially twice to ensure purity. For gel source data, see Supplementary Fig. 1. (c) NP-specific IgG+ PCs in the spleen of the indicated mice. All mice were given tamoxifen-laced chow for 4 weeks beginning at 5 weeks of age, then immunized with NP-KLH/alum and analysed by ELISPOT 30 days later. Error bars represent the mean (n = 5/grp). (d) NP-specific GC B cells in separate tamoxifen-fed WT;CD20-TAM-Cre and P2rX4f/f; CD20-TAM-Cre adults immunized 14 days previously. Graph shows means for hapten-binding GC B cells for 3 mice/group. For a comprehensive illustration of parent gates for evaluating NP-specific GC B cells see Extended Data Fig. 3. Experiments in every panel including the PCR data in (b) were performed twice. All p values were derived from two-tailed Mann–Whitney tests for all plots, without any adjustment for multiple comparisons.
Extended Data Fig. 8 Differential impact of P2rX4 inhibition on BM PCs versus naïve and GC B cells.
Flow cytometric analysis of BM and spleen cells from B6 adult females given 5-BDBD (4.25 mg/kg) or DMSO alone as control every 2 days i.v. 4 times. All analyses occurred 8 days after the first dose. (a-c) Shown are steady-state B220+/− BM PCs (a), splenic PCs (b) and naïve splenic T and B cells and BM B-lineage cells (c) from unimmunized mice. For panels (a-c) n = 6/grp from one of three experiments. (d) NP-specific GC B cells in separate mice immunized 14 days before with NP-KLH (n = 3/grp, representative of two separate experiments). For a comprehensive illustration of parent gates for evaluating NP-specific GC B cells see Extended Data Fig. 3. (e) Numbers and representative flow cytometric analysis of peritoneal cavity CD19+ IgM+ CD43+ B1 B cells in control and 5-BDBD treated C57BL/6 adults. n = 4/group. All bar graphs are means. All p values were derived from two-tailed Mann–Whitney tests for all plots, without any adjustment for multiple comparisons.
Extended Data Fig. 9 Rapid recovery of serum antibody titre and proteinuria following rapamycin.
NZB/W mice were monitored for serum dsDNA-specific IgG titre (a) and urine protein levels (b) over the indicated time frame. Protein scores were graded on a semiquantitative scale: 1, ≥30 mg/dl protein; 2, ≥100 mg/dl; 3, ≥300 mg/dl; and 4, ≥2,000 mg/dl. Twice weekly rapamycin (20 mg/kg) was administered i.v. from 29 to 33 weeks old age. Data represent means and are representative of 2 separate experiments each using 4 mice per group. Treatment windows are shown with grey rectangles.
Extended Data Fig. 10 Induced CHOP mutation in mature BM PCs.
Genomic DNA was prepared from sorted cells collected from CHOPf/f;CD20-TAM-Cre adults that were fed tamoxifen-laced chow for 4 weeks and evaluated the following day (n = 3). Sorted cell populations were mature BM PCs (CD138high Sca-1+ B220−) and B (surface IgM+) and T (CD3+) cells that were then subjected to PCR to amplify the CHOP (top) or HIF1α locus. For gel source data, see Supplementary Fig. 1.
Supplementary information
Supplementary Fig. 1
Uncropped gels for genomic PCR analyses.
Supplementary Table 1
List of oligonucleotides used for genomic PCR and qPCR analyses.
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Ishikawa, M., Hasanali, Z.S., Zhao, Y. et al. Bone marrow plasma cells require P2RX4 to sense extracellular ATP. Nature 626, 1102–1107 (2024). https://doi.org/10.1038/s41586-024-07047-2
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DOI: https://doi.org/10.1038/s41586-024-07047-2
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