Abstract
Germinal center (GC) B cells are crucial for the generation of GCs and long-lived humoral immunity. Here we report that one-carbon metabolism determines the formation and responses of GC B cells. Upon CD40 stimulation, GC B cells selectively upregulate methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) expression to generate purines and the antioxidant glutathione. MTHFD2 depletion reduces GC B cell frequency and antigen-specific antibody production. Moreover, supplementation with nucleotides and antioxidants suffices to promote GC B cell formation and function in vitro and in vivo through activation of the mammalian target of rapamycin complex 1 signaling pathway. Moreover, we found that antigen stimulation enhances YY1 binding to the Mthfd2 promoter and promotes MTHFD2 transcription. Interestingly, these findings can be generalized to the pentose phosphate pathway, which is another major source of reducing power and nucleotides. Therefore, these results suggest that an increased capacity for nucleotide synthesis and redox balance is required for GC B cell formation and responses, revealing a key aspect of GC B cell fate determination.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on SpringerLink
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Data availability
All data supporting the findings of this study are included in the article and the Supplementary Information. Source data are provided with this paper.
References
Victora, G. D. & Nussenzweig, M. C. Germinal centers. Annu Rev. Immunol. 30, 429–457 (2012).
Klein, U. & Dalla-Favera, R. Germinal centres: role in B-cell physiology and malignancy. Nat. Rev. Immunol. 8, 22–33 (2008).
Victora, G. D. et al. Germinal center dynamics revealed by multiphoton microscopy with a photoactivatable fluorescent reporter. Cell 143, 592–605 (2010).
Jellusova, J. Metabolic control of B cell immune responses. Curr. Opin. Immunol. 63, 21–28 (2020).
Caro-Maldonado, A. et al. Metabolic reprogramming is required for antibody production that is suppressed in anergic but exaggerated in chronically BAFF-exposed B cells. J. Immunol. 192, 3626–3636 (2014).
Jayachandran, N. et al. TAPP adaptors control B cell metabolism by modulating the phosphatidylinositol 3-kinase signaling pathway: a novel regulatory circuit preventing autoimmunity. J. Immunol. 201, 406–416 (2018).
Diaz-Munoz, M. D. et al. The RNA-binding protein HuR is essential for the B cell antibody response. Nat. Immunol. 16, 415–425 (2015).
Dufort, F. J. et al. Glucose-dependent de novo lipogenesis in B lymphocytes: a requirement for ATP–citrate lyase in lipopolysaccharide-induced differentiation. J. Biol. Chem. 289, 7011–7024 (2014).
Cheng, J. et al. Fumarate suppresses B-cell activation and function through direct inactivation of LYN. Nat. Chem. Biol. 18, 954–962 (2022).
Cho, S. H. et al. Germinal centre hypoxia and regulation of antibody qualities by a hypoxia response system. Nature 537, 234–238 (2016).
Semenza, G. L., Roth, P. H., Fang, H. M. & Wang, G. L. Transcriptional regulation of genes encoding glycolytic enzymes by hypoxia-inducible factor 1. J. Biol. Chem. 269, 23757–23763 (1994).
Jellusova, J. et al. GSK3 is a metabolic checkpoint regulator in B cells. Nat. Immunol. 18, 303–312 (2017).
Sharma, R. et al. Distinct metabolic requirements regulate B cell activation and germinal center responses. Nat. Immunol. 24, 1358–1369 (2023).
Weisel, F. J. et al. Germinal center B cells selectively oxidize fatty acids for energy while conducting minimal glycolysis. Nat. Immunol. 21, 331–342 (2020).
Chen, D. et al. Coupled analysis of transcriptome and BCR mutations reveals role of OXPHOS in affinity maturation. Nat. Immunol. 22, 904–913 (2021).
Ducker, G. S. et al. Reversal of cytosolic one-carbon flux compensates for loss of the mitochondrial folate pathway. Cell Metab. 23, 1140–1153 (2016).
Galibert, L. et al. CD40 and B cell antigen receptor dual triggering of resting B lymphocytes turns on a partial germinal center phenotype. J. Exp. Med. 183, 77–85 (1996).
D’Avola, A. et al. PHGDH is required for germinal center formation and is a therapeutic target in MYC-driven lymphoma. J. Clin. Invest. 132, e153436 (2022).
Haniuda, K., Nojima, T. & Kitamura, D. In vitro-induced germinal center B cell culture system. Methods Mol. Biol. 1623, 125–133 (2017).
Kawai, J. et al. Structure-based design and synthesis of an isozyme-selective MTHFD2 inhibitor with a tricyclic coumarin scaffold. ACS Med. Chem. Lett. 10, 893–898 (2019).
Li, G., Wu, J., Li, L. & Jiang, P. p53 deficiency induces MTHFD2 transcription to promote cell proliferation and restrain DNA damage. Proc. Natl Acad. Sci. USA 118, e2019822118 (2021).
Gustafsson Sheppard, N. et al. The folate-coupled enzyme MTHFD2 is a nuclear protein and promotes cell proliferation. Sci. Rep. 5, 15029 (2015).
Nojima, T. et al. In-vitro derived germinal centre B cells differentially generate memory B or plasma cells in vivo. Nat. Commun. 2, 465 (2011).
Zhao, R. et al. A GPR174–CCL21 module imparts sexual dimorphism to humoral immunity. Nature 577, 416–420 (2020).
Emmanuel, N. et al. Purine nucleotide availability regulates mTORC1 activity through the Rheb GTPase. Cell Rep. 19, 2665–2680 (2017).
Jiang, P., Du, W. & Wu, M. Regulation of the pentose phosphate pathway in cancer. Protein Cell 5, 592–602 (2014).
Patra, K. C. & Hay, N. The pentose phosphate pathway and cancer. Trends Biochem. Sci. 39, 347–354 (2014).
Franchina, D. G. et al. Glutathione-dependent redox balance characterizes the distinct metabolic properties of follicular and marginal zone B cells. Nat. Commun. 13, 1789 (2022).
Wang, L. W. et al. Epstein–Barr-virus-induced one-carbon metabolism drives B cell transformation. Cell Metab. 30, 539–555 (2019).
Acknowledgements
We thank H. Qi, M. Xu, W. Liu, L. Yu and D. Pan at Tsinghua University for materials and/or technical assistance. We thank all members of the Jiang laboratory for their technical assistance and/or discussions. We thank X. Liu, L. Xu, X. Wang and W. Wang for their help with the LC–MS/MS experiments. This work was supported by the National Natural Science Foundation of China (82125030 and 82341022 to P.J.) and the National Natural Science Foundation of China (82273227 to J.W.). J.W. was also supported by the Shuimu Tsinghua Scholar Program.
Author information
Authors and Affiliations
Contributions
J.W., G.L. and P.J. designed the experiments. J.W. and J.Z. performed all of the experiments except those mentioned below. G.L. performed some of the animal and imaging experiments. X.S., X.C. and H.C. provided technical assistance. J.W. collected, analyzed and organized the data with help from J.Z. P.J. supervised the research and interpreted the data. P.J. wrote the paper with the help of W.J. and J.Z. All authors commented on the paper.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Peer review
Peer review information
Nature Chemical Biology thanks the anonymous reviewer(s) for their contribution to the peer review of this work.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data
Extended Data Fig. 1 GC B cells display increased one-carbon metabolism activity.
a, Schematic illustration of carbon transition from serine into purines through one-carbon metabolism catalyzed by SHMT2 and MTHFD2. Red circle represents 13C from serine. b, Mass isotopomer distribution (MID) of AMP, GMP, CMP and UMP in B cells cultured in medium containing 13C3-serine after 24 or 48 hours of activation with anti-CD40+IgM+IgG F(ab)2. Cells per group from n = 3 mice. c-f, Germinal center B cells, follicular B cells, marginal zone B cells and transitional B cells were isolated from splenocytes of 7-day NP-KLH-immunized C57BL/6 mice, and splenocytes or splenocytes pre-treated for 24 h with GSH or NAC were analyzed for glutathione levels by LC-MS (c), or ROS levels by DCF staining (d) and survival by PI staining (e) followed by FACS analysis. FACS plots are shown (f). n = 3 mice per group. All data are the mean ± SEM.; Two-tailed Student’s t-test (c-e); ns, not significant.
Extended Data Fig. 2 Serine supports T cell-dependent GC B cell differentiation.
a,b, Levels of 13C-labled glycine (a), or methionine, CMP, TMP and UMP (b) in naive B cells activated by anti-CD40+IgG+IgM F(ab)2 in medium containing 400 µM 13C3-serine for the indicated time points. Cells per group from n = 3 mice c, d, Mouse naïve B cells activated by anti-CD40 + mIL4 for 72 hours in medium with (+) or without (-) serine and glycine (Serine/Glycine, SG) were stained with CFSE (c) or Ki67 antibody (d) and analyzed by FACS. Cells per group from n = 3 mice. e, f, Male C57BL/6 mice (6 weeks old) fed on serine/glycine-free diet were intravenously injected with PBS (-SG) or Serine/Glycine ( + SG) every 2 days, and immunized with or without (-) NP-KLH (e) or NP-LPS (f) on day 7 for another 7 days. The percentage of GC B cells and NP+ GC B cells were analyzed by FACS. n = 6 mice per group (-SG or +SG) or 3 mice for untreated (-) group. All data are the mean ± SEM.; Two-tailed Student’s t-test (d-f); ns, not significant.
Extended Data Fig. 3 B cell-specific knockout MTHFD2 reduces GC B-cell number and antigen-specific antibody production.
a-e, Splenocytes and serum were collected from female Mthfd2+/+ and Mthfd2−/− mice (n = 6 per group) immunized with NP-KLH on days 7 and 14 respectively. (a) Scheme of the experiments. The percentage of plasma cells relative to spleen cells (b), GC B cells relative to total B cells (c), or NP+ GC B cells of GC B cells (d) were analyzed by FACS. Serum titers of high-affinity (NP7) (f) and total (NP25) (e) IgG1, IgG2a, IgG2b, IgG3 and IgM at 14 days post immunization were also measured. Data are the mean ± SEM.; Two-tailed Student’s t-test (b-f); ns, not significant.
Extended Data Fig. 4 MTHFD2-mediated metabolic alteration modulates GC B-cell formation and responses.
a,b, Levels of the indicated metabolites in B cells treated as in Fig. 3a. Cells per group from n = 3 mice. c, Levels of the indicated metabolites in activated B cells treated with or without DS44. Cells per group from n = 3 mice. d, 13C-glutathione abundance in naive B cells activated for the indicated time points in medium containing 13C3-serine. Cells per group from n = 3 mice. e, ROS levels in each cell type from spleens of NP-KLH-immunized mice (n = 3 per group). All data are the mean ± SEM.; Two-tailed Student’s t-test (c-e); ns, not significant.
Extended Data Fig. 5 Effects of purines and GSH on IgG+ class switching in B cells.
a, Class switching of naive B cells activated by anti-CD40 + mIL4 for 72 h in the medium with different doses of purines and/or GSH as indicated. Cells per group from n = 3 mice. b, c, Relative to Extended Data Fig. 5a. Cell proliferation and survival were detected by CFSE staining (b) and PI staining (c) respectively. d, Surface expression of GL7 and Fas on iGBs generated from Mthfd2+/+ and Mthfd2−/− B cells (n = 3 independent wells per treatment). e,f, Naive B cells were activated with anti-CD40 + mIL4 in the medium containing 200 μM adenosine monophosphate-13C10, 15N5 or 200 μM Guanosine-5 ‘- monophosphate-15N5 sodium for 24 h. The levels of labelled indicated metabolites in B cells were analyzed. Cells per group from n = 3 mice. All data are the mean ± SEM.; Two-tailed Student’s t-test (a, c, d); ns, not significant.
Extended Data Fig. 6 Supplementation of nucleotides and GSH sufficiently promotes GC B-cell formation and antigen-specific antibody production in response to NP-KLH or sheep blood red cells (SRBCs) antigen challenge in vivo.
a, Male Mthfd2+/+ and Mthfd2−/− mice (6 weeks old) (n = 6 per group) intraperitoneally injected with PBS (-) or purines and/or GSH every 2 days for 7 days were then immunized with NP-KLH. the serum was collected on day 21 and serum titers of total (NP25) of IgG2a and IgG2b, and high-affinity (NP7) of IgG2b were measured. b-e, Male Mthfd2+/+ and Mthfd2−/− mice (6 weeks old) (n = 6 per group) intraperitoneally injected (i.p.) with PBS, 100 mM purines plus 200 mM GSH every 2 days for 14 days were then immunized with 5×108 cells/mouse sheep blood red cells (SRBCs) by intraperitoneal injection (b). On day 14, spleens were collected and the percentage of GC B cells (Fas+GL7+, pregated on B220+ cells) were analyzed by FACS (c). The serum was collected on day21 and the relative titers of SRBCs-specific IgG1, IgM, IgG3, IgG2a and IgG2b were measured (d, e). All data are the mean ± SEM.; Two-tailed Student’s t-test (c-e); ns, not significant.
Extended Data Fig. 7 Adenosine, guanosine and GSH suffice to form GC B cells.
a,b, Male Mthfd2+/+ and Mthfd2−/− mice (6-7 weeks old, n = 6 per group) were injected intraperitoneally every 2 days with DMSO (Ctrl), or 100 mM Ado and Guo (20 μl/mouse) plus 100 mM GSH (100 μl/mouse) until the end of the experiment. Mice were immunized with NP-KLH for 7 days and half, and the percentage of splenic NP+ GC B cells was measured (a). After a further 7 days, the splenic memory B cells were measured by FACS (b). All data are the mean ± SEM.; Two-tailed Student’s t-test; ns, not significant.
Extended Data Fig. 8 YY1 Transcriptional upregulation of MTHFD2 in GC B cells.
a, NIH3T3 cells transiently transfected with the indicated expression plasmids were analyzed by qPCR and western blot, respectively. n = 3 independent wells for each treatment. b,d, Western blot analysis of activated B cells transfected with shVector, shATF4 (b) or shMYC (d) for 48 h. c,e, Activated B cells transfected with MIG-GFP vector or MIG-GFP-ATF4 (c) or MIG-GFP-MYC (e) for 48 h were analyzed by Western blot. f,g, ChIP assay for binding of YY1 to the response element 2 in the Mthfd2 promoter (YY1 RE2) using anti-nonspecific IgG or anti-YY1 antibody in mouse primary B cells without stimulation or stimulated by CD40 and IL4 (f), or in isolated mouse non-GC or GC cells (g). Bound DNA was amplified by PCR using primers for RE2 or actin. Expression of YY1 and Actin was analyzed by Western blot. h, Proliferation and survival of activated B cells from Mthfd2+/+ and Mthfd2−/− mice (n = 3 per group) transfected with shVector or shYY1 for 48 h. All data are the mean ± SEM.; Two-tailed Student’s t-test; Data in a-g are representative of three independent experiments.
Extended Data Fig. 9 PPP is upregulated in B cells upon CD40 and mIL4 stimulation.
a, Non-GC B cells and GC B cells from male NP-KLH-immunized mice (n = 3) were analyzed by Western blot. b, Naive B cells activated for the indicated time points were analyzed by Western blot. c, Schematic diagram of the pentose phosphate pathway (PPP). d-f, Naive B cells were incubated with 11.1 mM 13C6-Glucose, or activated in the presence of 11.1 mM 13C6-Glucose in glucose-free DMEM as indicated. The levels of 13C-labelled glucose 6-phosphate(d), ribose 5-phosphate(e), 5-phosphoribosyl 1-pyrophosphate, IMP and AMP (f) were analyzed by LC-MS. Cells per group from n = 3 mice. g, Mouse naive B cells were activated in the absence (Ctrl) or presence of 6-Aminonicotinamide (6-AN), GSH and purines (AMP, GMP, CMP and TMP, referred to as A/T/C/G) as indicated. The levels of the metabolites were measured. Cells per group from n = 3 mice. All data are the mean ± SEM.; Two-tailed Student’s t-test (g); ns, not significant. Data in a and b are representative of three independent experiments.
Supplementary information
Supplementary Information
Supplementary Figs. 1–14 and image source data for supplementary figures.
Supplementary Table 1
Lists of the antibodies and primers used in this study.
Supplementary Table 2
Statistical source data for supplementary figures.
Source data
Source Data Fig. 1
Unprocessed western blots and/or gels.
Source Data Fig. 1
Statistical source data.
Source Data Fig. 2
Unprocessed western blots and/or gels.
Source Data Fig. 2
Statistical source data.
Source Data Fig. 3
Statistical source data.
Source Data Fig. 4
Unprocessed western blots and/or gels.
Source Data Fig. 4
Statistical source data.
Source Data Fig. 5
Unprocessed western blots and/or gels.
Source Data Fig. 5
Statistical source data.
Source Data Fig. 6
Statistical source data.
Source Data Extended Data Fig. 1
Statistical source data.
Source Data Extended Data Fig. 2
Statistical source data.
Source Data Extended Data Fig. 3
Statistical source data.
Source Data Extended Data Fig. 4
Statistical source data.
Source Data Extended Data Fig. 5
Statistical source data.
Source Data Extended Data Fig. 6
Statistical source data.
Source Data Extended Data Fig. 7
Statistical source data.
Source Data Extended Data Fig. 8
Unprocessed western blots and/or gels.
Source Data Extended Data Fig. 8
Statistical source data.
Source Data Extended Data Fig. 9
Unprocessed western blots and/or gels.
Source Data Extended Data Fig. 9
Statistical source data.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Wu, J., Zhou, J., Li, G. et al. Metabolic determinants of germinal center B cell formation and responses. Nat Chem Biol (2024). https://doi.org/10.1038/s41589-024-01690-6
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41589-024-01690-6
This article is cited by
-
One-carbon footprint in B cells
Nature Chemical Biology (2024)
