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Gsk3 is a metabolic checkpoint regulator in B cells

Abstract

B cells predominate in a quiescent state until an antigen is encountered, which results in rapid growth, proliferation and differentiation of the B cells. These distinct cell states are probably accompanied by differing metabolic needs, yet little is known about the metabolic control of B cell fate. Here we show that glycogen synthase kinase 3 (Gsk3) is a metabolic sensor that promotes the survival of naive recirculating B cells by restricting cell mass accumulation. In antigen-driven responses, Gsk3 was selectively required for regulation of B cell size, mitochondrial biogenesis, glycolysis and production of reactive oxygen species (ROS), in a manner mediated by the co-stimulatory receptor CD40. Gsk3 was required to prevent metabolic collapse and ROS-induced apoptosis after glucose became limiting, functioning in part by repressing growth dependent on the myelocytomatosis oncoprotein c-Myc. Notably, we found that Gsk3 was required for the generation and maintenance of germinal center B cells, which require high glycolytic activity to support growth and proliferation in a hypoxic microenvironment.

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Figure 1: GC B cells face increased metabolic demands.
Figure 2: Gsk3 promotes B cell quiescence and homeostasis.
Figure 3: Gsk3 is required for T cell–dependent B cell responses.
Figure 4: Gsk3 inhibits CD40-induced B cell proliferation.
Figure 5: Gsk3 limits CD40-induced metabolic activity.
Figure 6: Gsk3 promotes rapamycin sensitivity and c-Myc degradation.
Figure 7: c-Myc as a functional target of Gsk3.
Figure 8: Gsk3 promotes B cell survival under glucose restriction.

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Acknowledgements

We thank the SBP vivarium staff for animal care, M. Shlomchik (University of Pittsburgh) for providing the hCD20-TamCre mice, D. Kitamura (Tokyo University of Science) for providing the CD40LB cell line and C. Lyssiotis (University of Michigan) for discussions. Supported by US National Institutes of Health grant R01AI41649 (R.C.R.), the Lilly Research Award Program (R.C.R.), fellowships from the Deutsche Forschungsgemeinschaft (J.J.) and the Cancer Centers Council (C3) (P.R.-R.) and grants from the Arthritis National Research Foundation (J.J.) and the Canadian Institutes of Health Research (J.W.). The Animal Resources and Cancer Metabolism Cores at SBP are supported by NCI award 5P30CA030199.

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Authors and Affiliations

Authors

Contributions

J.J. designed and performed the majority of the experiments, analyzed the data and, together with R.C.R., wrote the manuscript; M.H.C. performed and analyzed the NP–KLH immunization experiment and contributed to the initial phenotypic analysis of Gsk3-deficient mice; J.R.A. performed and analyzed the CyTOF experiments and annexin V stainings; P.R.-R. performed and analyzed the in vivo CD40 stimulations and contributed to the experiments analyzing B cell proliferation in vivo and in vitro; C.R.L. and C.C. provided technical assistance with the experiments; A.D.R. helped perform and interpret the analysis of B cell metabolism; E.M.C. and R.J.B. provided advice, resources and assistance with the CyTOF experiments; J.R.W. provided mice and conceptual input to the manuscript; and R.C.R. conceived of and coordinated the study, interpreted the data and wrote the manuscript.

Corresponding author

Correspondence to Robert C Rickert.

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Competing interests

E.M.C. and R.J.B. are paid employees of Eli Lilly and Company.

Integrated supplementary information

Supplementary Figure 1 Germinal center B cells face increased metabolic demands in a hypoxic environment.

(a-c) Mice were immunized with SRBC, injected with PBS (n=11 mice) or 2DG (n=9 mice) on day 4,5,6 and analyzed on day 7. The percentage of B220+ cells (a), ratio of CD4+/CD8+ cells (b) and the frequency of PD1+ cells (c) in the spleen are shown. (d) Phosphorylation of the signaling molecules BLNK (Y84), Gsk3β (S9), Erk (T202/Y204), S6 (S235/236) and PLCγ2 (Y759) in wild type marginal zone B cells (B220+, CD21hi, IgMhi, CD23lo) at the indicated time points after stimulation with anti-IgM were analyzed by CyTOF Mass Cytometry. (e) c-Myc and β-Catenin expression and GSK3β phosphorylation (S9) in wild-type GC B cells (B220+, CD19+, GL7+, Fas+) were analyzed by CyTOF Mass Cytometry. Data are displayed using the t-Distributed Stochastic Neighbor Embedding (tSNE) algorithm. Horizontal line represents the mean (a, b, c). Data are representative of three (a, b, c) and one (d, e) independent experiments.

Supplementary Figure 2 Gsk3 promotes peripheral B cell quiescence and homeostasis.

(a+b) Total numbers of follicular B cells in the spleen (B220+, CD23+, CD21lo) (n=12 Ctrl mice and 13 dKO mice) (b) and B cells in lymph nodes (B220+) (n=10 Ctrl mice and 7 dKO mice) Mice used: Cd19Cre (Ctrl) and Gsk3aL/L Gsk3bL/LCd19Cre (dKO) (c) Analysis of frozen spleen sections by histology. Scale bar shows 100μm. Antibodies specific for: B220 or IgM were used to detect B cells, Moma1 to detect metallophilic macrophages, CD5 to detect T cells, and CD35 to detect follicular dendritic cells. Mice used: Cd19Cre (Ctrl) and Gsk3aL/LGsk3bL/LCd19Cre (dKO). (d) Flow cytometric analysis of B cell development in the bone marrow. First row shows all live cells in the bone marrow, cells in the second row are pre-gated as: B220+ and CD43-, cells in the third row are pre-gated as: B220+, CD43+. Plots are representative of 3 mice per genotype. Mice shown: Cd19Cre (Ctrl) and Gsk3aL/LGsk3bL/LCd19Cre (dKO) (e) Histograms show IgD expression and IgM expression on follicular B cells (B220+, CD23+, CD21-). Plots are representative of 9 mice per genotype. Mice shown: Cd19Cre (Ctrl) and Gsk3aL/LGsk3bL/LCd19Cre (dKO) (f) Relative frequency of YFP+ splenic marginal zone B cells from hCd20-TamCre (Ctrl) and Gsk3aL/LGsk3bL/LhCd20-TamCre (dKO) mice 10, 26 and 47 days after tamoxifen injection. The measured frequency of YFP+ cells was normalized to the value obtained in the blood at the peak of induction (d7) Horizontal line represents the mean (a, b, f). Data are representative of thirteen (a), five (b), one (c), three (d), nine (e) and two (f) independent experiments. *P=0.0172, *P=0.0268 t test (a, b), *P=0.0181, **P=0.0061 ANOVA (f) and *P=0.0121 Mann Whitney test (f).

Supplementary Figure 3 Gsk3 inhibits CD40-induced B cell proliferation.

(a) Histograms show proliferation of Ctrl and dKO B cells after 3 days of cell culture treated with the indicated amount of anti-CD40. One representative mouse out of four per genotype is shown. (b) Cells were stimulated with anti-IgM for 16h and subsequently with anti-CD40+IL-4+BAFF for 3 days. Histograms show proliferation of Ctrl and dKO B cells on the last day of the experiment. One representative mouse out of four per genotype is shown. (c) B cells from Ctrl and dKO mice were cultured as described in (b), cells size was determined using FSC. Mice used: Gsk3aL/L x Gsk3bL/L x hCd20-TamCre- (Ctrl) Gsk3aL/L x Gsk3bL/L x hCd20-TamCre (dKO). All mice were injected with tamoxifen on 3 consecutive days. Horizontal line represents the mean (c). Data are representative of one (a, b, c) experiment. P=0.0857Mann Whitney test (c).

Supplementary Figure 4 Gsk3 is dispensable for anti-IgM-induced metabolic adaptations.

Basal OCR (left) and ECAR (right) levels in B cells stimulated over night with anti-IgM are shown. Graphs summarize data obtained from 3 independent experiments with 8 mice per genotype in total. For each of the three experiments, the values were normalized to a value obtained from one random wildtype sample. Mice used: hCd20-TamCre or Gsk3aL/LGsk3bL/L (Ctrl) Gsk3aL/LGsk3bL/LhCd20-TamCre (dKO) All mice were injected with tamoxifen on 3 consecutive days. Horizontal line represents the mean.

Supplementary Figure 5 Gsk3 promotes c-Myc degradation.

(a) Expression of the indicated proteins in freshly isolated B cells and B cells cultured over night with anti-CD40 and IL4 from Ctrl and dKO mice was analyzed by western blot. Gsk3aL/LGsk3bL/L (Ctrl) Gsk3aL/LGsk3bL/LhCd20-TamCre (dKO). All mice were injected with tamoxifen on 3 consecutive days. (b+c) B cells from Ctrl and dKO mice were cultured with anti-CD40+IL4 (b) or anti-IgM over night (c) and c-Myc expression was analyzed by western blot. Shown are ratios of c-Myc band intensities and the band intensity of the respective loading control. Mice used: hCd20-TamCre or Gsk3aL/LGsk3bL/L (Ctrl) Gsk3aL/LGsk3bL/LhCd20-TamCre(dKO). All mice were treated with tamoxifen on 3 consecutive days. Data are representative of one (a), ten (b) and three (c) independent experiments. **P=0.0095 paired t test (a).

Supplementary Figure 6 Normal B cell development in Myc-tg mice.

Analysis of B cell maturation in the spleen from R26StopFLMycCd19cre mice (Myc-Tg) and Cd19Cre littermates (Ctrl). Plots are representative for 5 mice per genotype analyzed in 5 independent experiments.

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Jellusova, J., Cato, M., Apgar, J. et al. Gsk3 is a metabolic checkpoint regulator in B cells. Nat Immunol 18, 303–312 (2017). https://doi.org/10.1038/ni.3664

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