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.

Figure 1. GC B cells face increased metabolic demands

(a) Cell size (FSC-A) of freshly isolated GC B cells (n=10) (B220⁺, Fas⁺, GL7⁺) and non-GC B cells (n=10) (B220⁺, Fas⁻, GL7⁻). Significance (****p<0.0001) was determined using the Mann-Whitney test (b) Protein levels of GC B cells (n=6) (B220⁺, Fas⁺, GL7⁺) and non-GC B cells (n=6) (B220⁺, Fas⁻, GL7⁻) B cells. Significance (p=0.06) was determined using the Mann-Whitney test (c) Mice were immunized with SRBC and i.v.-injected with 2NBDG 7 days later. 2NBDG uptake was measured by flow cytometry. The obtained MFIs from GC B cells (n=6) (B220⁺, Fas⁺, GL7⁺) and non-GC B cells (n=6) (B220⁺, Fas⁻, GL7⁻) are shown. Significance (**p=0.002) was determined using the Mann-Whitney test (d) Mitochondrial mass as determined by MitoTracker Red CMX Ros labeling of GC and non GC B cells. Significance (****p<0.0001) was determined using the Mann-Whitney test (e) Detection of hypoxic regions in the spleen from SRBC immunized mice. Results are representative for 3 mice. Scale bar shows 10 µ1. (f) Hif-1α expression in lysates from purified GC B cells (Fas⁺, GL7⁺, B220⁺, CD11c⁻, CD43⁻, IgD⁻) and non-GC B cells (Fas⁺, GL7⁺, B220⁺, CD11c⁻, CD43⁻, GL7⁻). Results are representative of 4 sets of samples. (g) Mice were immunized with SRBC, injected with PBS (n=11) or 2DG (n=9) on day 4,5,6 and sacrificed on day 7. The percentage of GC B cells (B220⁺, Fas⁺, GL7⁺) in the spleen is shown. Significance (****p<0.0001) was determined using the t-test with Welch‘s correction. (h) Phosphorylation and expression of the indicated molecules in GC B cells (B220⁺, CD19⁺, GL7⁺, Fas⁺) was analyzed by CyTOF Mass Cytometry. Numbers highlighted in red show mean values obtained from non-GC B cells. Data are displayed using the t-Distributed Stochastic Neighbor Embedding (tSNE) algorithm. P-BLNK, p-GSK3, p-Erk, pS6 and p- PLCγ2 levels were used as sorting parameters. Plots are representative of 4 independent experiments. All experiments were performed using wildtype mice. Mice were sacrificed 7 days after immunization.

Figure 2. GSK3 promotes B cell quiescence and homeostasis

(a+b) Graph shows the frequency of B cells (B220⁺) in the spleen (n=19 WT, 12 dKO) (a) and peripheral lymph nodes (n=10 WT, n=7 dKO) (b). Significance (for spleen **p=0.0021, for LN ***p=0.004) was determined using the t test and the Mann-Whitney test respectively. (c) Analysis of B cell maturation in the spleen. Displayed cells are pre-gated based on B220 expression. Plots are representative of >9 mice. Significance was determined using the t test (for values presented in the upper and middle panel) and the Mann-Whitney test (for values in the bottom panel) (**** p < 0.0001, ***p<0.001, **p<0.01) Mice used: Gsk3a^+/+ x Gsk3b^+/+ x Cd19^Cre (Ctrl) and Gsk3a^L/L x Gsk3b^L/L x Cd19^Cre (dKO). (d) GSK3 protein expression in lysates from splenic B cells isolated from Gsk3a^+/+ x Gsk3b^+/+ x Cd19^Cre (Ctrl), Gsk3a^L/L x Gsk3b^+/+ x Cd19^Cre (Gsk3a^L/L), Gsk3a^+/+ x Gsk3b^L/L x Cd19^Cre (Gsk3b^L/L) and Gsk3a^L/L x Gsk3b^L/L x Cd19^Cre (dKO) mice. MEK1/2 was used as loading control. Shown western blot is representative for 4 experiments. (e) Forward scatter (FSC-A) values as a measure of cell size in freshly isolated follicular (n=6) (B220⁺, CD23⁺, CD21^lo) and marginal zone (n=6) (B220⁺, CD23⁻, CD21^hi) B cells from Gsk3a^+/+ x Gsk3b^+/+ x Cd19^Cre (Ctrl), Gsk3a^L/L x Gsk3b^L/L x Cd19^Cre (dKO) mice. Significance (**p=0.002) was determined using the Mann-Whitney test. (f) Protein levels of follicular (n=6) (B220⁺, CD23⁺, CD21^lo) and marginal zone (n=6) (B220⁺, CD23⁻, CD21^hi) B cells from Gsk3a^+/+ x Gsk3b^+/+ x Cd19^Cre (Ctrl), Gsk3a^L/L x Gsk3b^L/L x Cd19^Cre (dKO) mice. Significance (**p=0.0043) was determined using the Mann-Whitney test. (g) Glucose uptake by follicular B cells (n=3) and marginal zone (n=3) B cells from Gsk3a^+/+ x Gsk3b^+/+ x Cd19^Cre (Ctrl), Gsk3a^L/L x Gsk3b^L/L x Cd19^Cre (dKO) mice injected with 2NBDG and analyzed 1h later by flow cytometry. One of two experiments with a total of 6 mice per genotype is shown. (h) Immunoblots for GSK3 protein from splenic B cells isolated from Gsk3a^+/+ x Gsk3b^+/+ x hCd20- Tam^Cre (Ctrl), Gsk3a^L/L x Gsk3b^+/+ x hCd20-Tam^Cre (Gsk3a^L/L), Gsk3a^+/+ x Gsk3b^L/L x hCd20-Tam^Cre (Gsk3b^L/L) and Gsk3a^L/L x Gsk3b^L/L x hCd20-Tam^Cre (dKO) mice. Mice were injected with tamoxifen on 3 subsequent days and analyzed 8 days after the last injection. MEK1/2 was used as loading control. (i) Relative frequency of YFP⁺ splenic follicular B cells from Gsk3a^+/+ x Gsk3b^+/+ x hCd20- Tam^Cre (Ctrl) and Gsk3a^L/L x Gsk3b^L/L x hCd20-Tam^Cre (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). Significance (*p=0.0121) was determined using the Mann-Whitney test. (j) FSC-A values of YFP⁺ B cells from the blood of Gsk3a^+/+ x Gsk3b^+/+ x hCd20- Tam^Cre (Ctrl) and Gsk3a^L/L x Gsk3b^L/L x hCd20-Tam^Cre (dKO) mice at day 0, 7, 14 and 20 after tamoxifen injection. Significance (*p=0.0357) was determined using the Mann-Whitney test.

Figure 3. GSK3 is required for T cell-dependent B cell responses

(a) Serum levels of SRBC-specific IgM (left) and IgG1 (right) antibody on d0 and d7 as measured by flow cytometric staining (WT n=11, dKO n=12). Plots show the obtained mean fluorescence intensities. Significance (****p<0.0001) was determined using the t test with Welch‘s correction (for IgM) and the Mann-Whitney test (for IgG1). (b) The relative frequency of plasma cells (CD138⁺, B220^lo) in the spleen 7 days after SRBC immunization is shown. (c) Dot plots of GC B cells 7 days after immunization with SRBC. Plots are representative of 11 WT and 12 dKO mice. Significance (**p=0.0013) was determined using the t test with Welch‘s correction (d) Analysis of frozen spleen sections 7days after SRBC immunization. Scale bar shows µ1. Images are representative of two mice per genotype analyzed. (e) Serum titers of high affinity (NP₄) and total (NP₂₃) IgM and IgG at days 0, 7 and 14 following NP-KLH immunization. Mice shown: Gsk3a^+/+ x Gsk3b^+/+ x hCd20-Tam^Cre or Gsk3a^L/L x Gsk3b^L/L x hCd20-Tam^Cre− (Ctrl) Gsk3a^L/L x Gsk3b^L/L x hCd20-Tam^Cre (dKO). Significance was determined using the two-way ANOVA test.

Figure 4. GSK3 inhibits CD40-induced B cell proliferation

(a) Induced germinal center B cells (iGB) were generated from Ctrl and dKO B cells using the feeder cell line CD40LB and exogenous IL-4. GL7 and Fas expression on iGB cells is shown. Plots are representative of 3 mice per genotype. (b) Histograms show B cell proliferation and (c) average number of cell divisions of Ctrl and dKO after 3 days of cell culture with the indicated stimuli. Mean values obtained from 3–7 experiments are shown. Significance (**p=0.0093) was determined using the Mann-Whitney test. (d) Mice were injected with anti-CD40, B cell proliferation was measured by BrdU incorporation 3 days later. Histograms show representative BrdU staining, the dot plot summarized the results from two independent experiments. Significance (**p=0.0043) was determined using the Mann-Whitney test. (e) FSC-A values of B cells from Ctrl and dKO mice cultured overnight with the indicated stimulations. Significance (**p=0.0079) was determined using the Mann-Whitney test.. Mice shown in a,b,c and e: Gsk3a^+/+ x Gsk3b^+/+ x hCd20-Tam^Cre or Gsk3a^L/L x Gsk3b^L/L x hCd20-Tam^Cre− (Ctrl) Gsk3a^L/L x Gsk3b^L/L x hCd20-Tam^Cre (dKO). Mice shown in d: Gsk3a^+/+ x Gsk3b^+/+ x Cd19^Cre or Gsk3a^L/L x Gsk3b^L/L x Cd19^Cre− (Ctrl) Gsk3a^L/L x Gsk3b^L/L x Cd19^Cre (dKO).

Figure 5. GSK3 limits CD40-induced metabolic activity

(a) Lactate and glucose concentrations in cell supernatants at day 3 of cell culture with the indicated stimuli is shown. B cells from tamoxifen-treated Ctrl and dKO mice were cultured at a concentration of 2x10⁶ cells/ml. Values obtained from medium without cells are shown for reference. Significance (*p=0.0159) was determined using the Mann-Whitney test (b) Oxygen consumption (OCR) and extracellular acidification (ECAR) of B cells isolated from tamoxifen-treated Ctrl and dKO mice and stimulated over night with anti-CD40⁺IL-4 is shown. The following inhibitors were used: A= Oligomycin, B=FCCP, C= Rotenone⁺Antimycin. Graphs are representative of 8 mice per genotype. (c) Basal OCR and ECAR levels in B cells stimulated over night with anti-CD40⁺IL-4 are shown. Graphs summarize data obtained from 3 independent experiments with 8 mice per genotype in total. In each experiment, values were normalized to a randomly chosen wildtype sample. Significance (**p=0.0036) was determined using the t test with the Welch‘s correction. Mice shown: Gsk3a^+/+ x Gsk3b^+/+ x hCd20-Tam^Cre or Gsk3a^L/L x Gsk3b^L/L x hCd20-Tam^Cre− (Ctrl) Gsk3a^L/L x Gsk3b^L/L x hCd20-Tam^Cre (dKO).

Figure 6. GSK3 promotes rapamycin sensitivity and c-Myc degradation

(a) Proliferation, (b) average number of cell divisions and (c) size (FSC-A) of Ctrl and dKO B cells after 3 days of cell culture with anti- CD40+IL-4 in the presence or absence of 25nM rapamycin. Significance (*p<0.05 **p=0.01,***p<0.001) was determined using the Mann Whitney test. (d) Proliferation, (e) average number of cell divisions and (f) size (FSC-A) of Ctrl and dKO B cells after 3 days of cell culture with anti-IgM in the presence or absence of 25nM rapamycin. Significance (**p=0.012 and 0.007 ***p=0.0003) was determined using the Mann Whitney test. (g) Cell lysates from Ctrl and dKO B cells stimulated with anti-CD40⁺IL-4 or (h) anti-IgM overnight in the presence or absence of 25nM rapamycin and probed for the indicated proteins. Results are representative of 3 independent experiments. Mice shown: Gsk3a^+/+ x Gsk3b^+/+ x hCd20-Tam^Cre or Gsk3a^L/L x Gsk3b^L/L x hCd20-Tam^Cre− (Ctrl) Gsk3a^L/L x Gsk3b^L/L x hCd20-Tam^Cre (dKO). All mice were treated with tamoxifen on 3 consecutive days.

Figure 7. c-Myc as a functional target of GSK3

(a) Myc protein levels in freshly isolated, anti-IgM or anti- CD40⁺IL-4 stimulated B cells from control and Myc-tg mice as determined by immunoblot. MEK1/2 was used as loading control. Results are representative of 3 independent experiments. (b) Cell size (FSC-A) of freshly isolated or (c) overnight cultured Ctrl and Myc-tg B cells. Significance (*p=0.0317) was determined using the Mann Whitney test. (d) Proliferation of Ctrl and Myc-tg B cells cultured for 3d with the indicated stimuli. Results are representative of 3 independent experiments. (e) Lactate (left) and glucose (right) concentration in cell supernatants at day 3 of cell culture with the indicated stimuli. Cells were cultured at a concentration of 1x10⁶ cells/ml. Values obtained in medium without cells is shown for reference. Significance (**p=0.0079) was determined using the Mann Whitney test. (f) Proliferation of Ctrl and Myc-tg B cells cultured over for 3 days with the indicated stimuli in the presence or absence of 25 nM rapamycin. Results are representative of 3 independent experiments. Mice used: R26Stop^FLMyc×Cd19^Cre (Myc-Tg) and Cd19^Cre littermates (Ctrl).

Figure 8. GSK3 promotes B cell survival under glucose restriction

(a) Percent viable Ctrl and dKO B cells at day 3 of cell culture with the indicated stimuli. Significance (p=0.013) was determined using the Mann Whitney test. (b) Percent GC B cells in spleens from Ctrl (n=13) and dKO (n=12) mice immunized with SRBC on d0, injected with tamoxifen on day 6,7,8 and analyzed on day 10. Mice shown: Gsk3a^+/+ x Gsk3b^+/+ x hCd20-Tam^Cre or Gsk3a^L/L x Gsk3b^L/L x hCd20-Tam^Cre− (Ctrl) Gsk3a^L/L x Gsk3b^L/L x hCd20-Tam^Cre (dKO). Significance (p<0.0001) was determined using the t test. (c) Percent Annexin V+ GC B cells in spleens from Ctrl (n=4) and dKO (n=4) mice immunized with SRBC on d0, injected with tamoxifen on day 5,6,7 and analyzed on day 8. Mice shown: Gsk3a^L/L x Gsk3b^L/L x hCd20-Tam^Cre− (Ctrl) Gsk3a^L/L x Gsk3b^L/L x hCd20-Tam^Cre (dKO). Significance (*p=0.0286) was determined using the Mann Whitney test. (d) Percent BrdU⁺ GC B cells in spleens from Ctrl (n=4) and dKO (n=4) mice immunized with SRBC on d0, injected with tamoxifen on day 5,6,7, injected with BrdU on day 9 and analyzed 3h later. Mice shown: Gsk3a^L/L x Gsk3b^L/L x hCd20-Tam^Cre− (Ctrl) Gsk3a^L/L x Gsk3b^L/L x hCd20-Tam^Cre (dKO).. Significance (p=0.0571) was determined using the Mann Whitney test. (e) Percent viable B cells from Gsk3a^+/+ x Gsk3b^+/+ x hCd20-Tam^Cre or Gsk3a^L/L x Gsk3b^L/L x hCd20-Tam^Cre− (Ctrl) Gsk3a^L/L x Gsk3b^L/L x hCd20-Tam^Cre (dKO). mice isolated from tamoxifen-treated mice and cultured for 3 days in complete or glucose-free medium, in the presence of anti-CD40+IL4 with or without 25 nM rapamycin or with or without NAC. Graph depicts 3 independent experiments. Significance (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001) was determined using the ANOVA test with Tukey‘s correction for multiple comparisons. (f) Mitochondrial mass of Gsk3a^+/+ x Gsk3b^+/+ x hCd20-Tam^Cre or Gsk3a^L/L x Gsk3b^L/L x hCd20-Tam^Cre− (Ctrl) Gsk3a^L/L x Gsk3b^L/L x hCd20-Tam^Cre (dKO).B cells isolated from tamoxifen-treated mice 10d after the last injection, cultured over night with the indicated stimuli. Significance (*p=0.0159, **p=0.0079) was determined using the Mann Whitney test. Values were from separate experiments were normalized to the value obtained from a randomly chosen unstimulated control sample. (g) Immunofluorescence of mitochondrial mass visualized by MitoTracker Red CMXRos staining in Gsk3a^+/+ x Gsk3b^+/+ x hCd20-Tam^Cre (Ctrl) Gsk3a^L/L x Gsk3b^L/L x hCd20-Tam^Cre (dKO). B cells stimulated with anti-CD40⁺IL4 over night. (h) Mitochondrial mass was measured with by MitoTracker Red CMXRos labeling of Ctrl and Myc-tg B cells cultured over night with the indicated stimuli. Values were from separate experiments were normalized to the value obtained from unstimulated control cells. Significance (**p=0.0079) was determined using the Mann Whitney test. (i) ROS production in anti-CD40⁺IL-4 stimulated Gsk3a^+/+ x Gsk3b^+/+ x Cd19^Cre (Ctrl) and Gsk3a^L/L x Gsk3b^L/L x Cd19^Cre (dKO) B cells cultured overnight in complete or glucose-free medium. Plots are representative for 3 independent experiments. (j) Freshly isolated splenic B cells from Gsk3a^L/L x Gsk3b^L/L x Cd19^Cre− or Gsk3a^+/+ x Gsk3b^+/+ x Cd19^Cre (Ctrl) and Gsk3a^L/L x Gsk3b^L/L x Cd19^Cre (dKO) mice were stained for ROS production. As a control, samples were stained in parallel for ROS content in the presence of the ROS scavenger NAC. Significance (**p=0.0025) was determined using the Mann Whitney test.