Stimulation with LPS + IL-4 promotes B cell proliferation and class switch recombination

(A-D) Cells from the lymph nodes of C57BL6/J mice were stained with Cell Trace Violet (CTV) and stimulated with IL-4 (10ng/ml) +/- LPS (20μg/ml) for 72 hours and analysed by flow cytometry. Data for naive B cells (stained on day 0) is also shown. Live CD19+ B cells were identified using the gating strategy described in Figure S14A. (A) Representative flow cytometry plots comparing IgG1 expression and CTV staining after 72 hours of IL-4 or LPS + IL-4 stimulation. (B) Representative plots for FSC and SSC at 72 hours of IL-4 or LPS + IL-4 stimulation with geometric means for the (C) forward scatter and (D) side scatter. Data shows the results of three biological replicates. Data was analysed by one-way ANOVA followed by multiple comparison testing via Sidak’s analysis. For comparisons to naïve B cells, p<0.0001 is indicated by ****. Full ANOVA results are given in Supplementary Table S5. (E-H) Cells from the lymph nodes of C57BL6/J mice, stimulated with LPS (20μg/ml) and IL-4 (10ng/ml) for 24 hours, and CD19+ B cells were isolated by FACS. Alternatively, naïve CD19+ B cells were sorted directly from ex vivo lymph node cells. Cells were lysed and analysed by proteomics as described in the methods. Samples from four mice for LPS + IL-4 and three for naive were generated. (E) Total protein content (pg/cell) was estimated from the proteomic data. Statistical power was determined using an unpaired two-tailed Student’s t-test, where p<0.01 is indicated by **. (F) Volcano plot depicting changes in estimated protein copy number in naïve vs LPS + IL-4 stimulated B cells. (G) Volcano plot showing the estimated cellular protein concentration (μM) in naïve vs LPS + IL-4 stimulated B cells. Horizontal dashed lines indicate q<0.05. Vertical dashed lines indicate log2 fold change of one standard deviation away from the median. (H) Enrichment analysis of the upregulated proteins in (G) against GO-term and KEGG databases.

Proteins involved in cell cycle progression are upregulated in LPS + IL-4 activated B cells

(A-B) B cells were purified from the spleens of C57BL6/J mice and either fixed on isolation (naive) or stimulated with LPS (20μg/ml) and IL-4 (10ng/ml) for 24 hours before fixation. Cells were then stained with DAPI and CD19. The cell cycle stages were analysed using the gating strategy shown in Figure S14B. (A) Representative histograms showing the proportion of B cells in different phases of the cell cycle. (B) Quantification shows three technical replicates from cells isolated from one mouse and is representative of three independent experiments. Statistical power was determined using two-way ANOVA followed by multiple comparison testing via Sidak’s analysis, where p<0.01 is indicated by **, p<0.001 by *** and p<0.0001 by **** for comparisons between the naive and LPS + IL-4 conditions. (C-F) Graphs depicting changes in the estimated cellular concentration (nM) of proteins implicated in entry into the cell cycle, determined from the proteomic dataset described in Figure 1. (C) Cyclin D, (D) CDK4, (E) CDK6, (F) p27Kip1. (G-H) B cells were purified from the spleens of C57BL6/J mice and stimulated with LPS (20μg/ml) and IL-4 (10ng/ml) for 24 hours before fixing and staining for phospho-retinoblastoma (p-Rb). Gating strategy for p-Rb staining is shown in Figure S14B. (G) Representative histogram comparing p-Rb staining in naïve and LPS + IL-4 stimulated B cells. (H) Quantification shows three technical replicates from cells isolated from one mouse and is representative of three independent experiments. (I) Heat map showing the expression of proteins encoded by E2F target genes, derived from the proteomic data. (J-M) Graphs depicting changes in the estimated cellular concentration (nM) of proteins implicated in cell cycle progression, determined from the proteomic dataset described in Figure 1. (J) Cyclin A, (K) Cyclin B, (L) CDK2, (M) CDK1. p values were determined using an unpaired two-tailed Student’s t-test for (H) or represent adjusted p values from the FDR calculations applied to the proteomic dataset (D, E, F, L), where p<0.01 is indicated by **, p<0.001 by *** and p<0.0001 by ****.

LPS + IL-4 stimulation promotes protein synthesis

(A, B) Graphs show changes in cellular concentration (nM) of the sum of proteins that make up the large (A) and small (B) ribosomal subunits, with heat maps showing the expression of the individual proteins making up the large (C) and small (D) subunits. (E-G) The proteomic dataset was mined for proteins involved in the biogenesis of the large and small ribosomal subunits based on the GO terms: GO:0000027, GO:0042273, GO:0000028, GO:0042274. (E) shows the sum of the proteins involved in the biogenesis of the large subunit and (F) shows the small subunit, with individual proteins represented on the volcano plot (G) Horizontal dashed lines on (G) indicate q < 0.05 while vertical dashed lines indicate log2 fold change more than one standard deviation away from the median. (H-I) Splenocytes from C57BL6/J mice were stimulated with LPS (20μg/ml) and IL-4 (10ng/ml) for 24 hours before fixing and staining for the uptake of puromycin analog O-propargyl-puromycin (OPP) to measure protein synthesis. Gating strategy for OPP staining is shown in Figure S14C. (H) Representative histogram comparing OPP uptake between naïve and LPS + IL-4 stimulated B cells. (I) Quantification shows three technical replicates from cells isolated from one mouse. Statistical power was determined using an unpaired two-tailed Student’s t-test, where p<0.001 is indicated by *** and p<0.0001 by ****.

Amino acid transporter SLC7A5 is required for key B cell functions

(A) Heat map showing the expression of genes encoding for proteins involved in plasma membrane amino acid transport determined from the proteomic dataset described in Figure 1. (B, C) Graphs depicting changes in cellular concentration (nM) of (B) SLC7A5 and (C) SLC3A2 derived from the proteomic data. Ap(adj) of <0.0001, based on the proteomic FDR calculations, is indicated by ****. (D-E) Lymph node cells from C57BL6/J mice were stimulated with LPS (20μg/ml) and IL-4 (10ng/ml) for 24 hours before staining for CD98. (D) Representative FACS plot comparing CD98 expression. (E) Quantification shows three technical replicates from cells isolated from one mouse and is representative of two independent experiments. Statistical power was determined using one-way ANOVA followed by multiple comparison testing via Dunnett’s analysis. For comparison to naïve B cells, where p<0.001 is indicated by *** and p<0.0001 by ****. (F-I) B cells were purified from the spleens of wild type (WT) and Slc7a5 fl/fl/Vav-iCre+ve mice and stained with CTV before stimulation with LPS (20μg/ml) and IL-4 (10ng/ml) for 72 hours. (F) Percentage of live B cells (7AAD-ve) in WT and Slc7a5fl/fl/Vav-iCre+vemice. (G) Histogram representing CTV staining of WT and Slc7a5fl/fl/Vav-iCre+ve B cells. (H) Live B cell number of WT and Slc7a5fl/fl/Vav-iCre+vemice. (I) Percentage of B cells that are IgG1+ve in WT and Slc7a5fl/fl/Vav-iCre+vemice. Data shows the results of four biological replicates per genotype. p<0.0001 is indicated by **** (unpaired two-tailed Student’s t-test). (J-K) B cells were purified from the spleens of WT and Slc7a5fl/fl/Vav-iCre+vemice and stimulated with LPS (20μg/ml) +/- IL-4 (10ng/ml) for 24 hours before fixing and staining for the uptake of kynurenine to measure amino acid uptake. Gating strategy for kynurenine uptake in Figure S14D. (J) Quantification of kynurenine MFI between B cells from WT and SLC7A5 KO mice with (+) or without (-) LPS + IL-4, kynurenine or aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH). (K) Quantification of kynurenine MFI between B cells from WT and Slc7a5fl/fl/Vav-iCre+vemice with (+) or without (-) LPS, kynurenine or BCH. Data shows the results of four biological replicates per genotype. Statistical power was determined for using two-way ANOVA followed by multiple comparison testing via Sidak’s analysis, where p<0.0001 is indicated by **** for comparisons between genotypes.

LPS + IL-4 stimulation upregulates cholesterol metabolism in B cells

(A-E) Analysis of the genes involved in cholesterol metabolism in the proteomic dataset. (A) Heat map of all the enzymes involved in cholesterol biosynthesis. Cellular concentration (nM) of (B) HMGCR, (C) SQLE, (D) LDLR, and (E). A p(adj) of <0.01 is indicated by **. (F) Splenocytes from C57BL6/J mice were plated in cholesterol-free (CF) media and stimulated with LPS (20μg/ml) and IL-4 (10ng/ml). The cells were fixed at the stated time points and stained with filipin. The gating strategy for filipin staining in Figure S15A. (F) Filipin timecourse. Data shows three technical replicates from cells isolated from one mouse. Statistical power was determined by one-way ANOVA followed by multiple comparison testing via Dunnett’s analysis. For comparisons to naïve B cells, p<0.05 is indicated by * and p<0.0001 by ****. (G-H) Splenocytes from C57BL6/J mice were plated in cholesterol-free media and pre-treated with DMSO as a vehicle control or varying concentrations of Fluvastatin or NB-598 for 45 minutes before stimulation with LPS (20μg/ml) and IL-4 (10ng/ml). The cells were fixed after 24 hours and stained with filipin before acquisition. (G) Filipin staining of Fluvastatin titration. (H) Filipin staining of NB-598 titration. Data shows three technical replicates from cells isolated from one mouse and is representative of two independent experiments, each with one biological replicate. Statistical power was determined using a one-way ANOVA followed by multiple comparison testing via Dunnett’s analysis. For comparison to LPS + IL-4 stimulated B cells, p<0.001 is indicated by *** and p<0.0001 by ****. (I) Spleenocytes from C57BL6/J mice were plated in normal or cholesterol-free (CF) media and pre-treated with DMSO as a vehicle control, Fluvastatin (10μM) or NB-598 (10μM) before stimulation with LPS + IL-4. The cells were fixed after 24 hours and stained with filipin before acquisition. Data shows the results of three biological replicates. Statistical power was determined using two-way ANOVA followed by multiple comparison testing via Tukey’s analysis. For comparisons to the LPS + IL-4 condition, p<0.0001 is indicated by ****.

Blocking rate-limiting enzymes in the cholesterol biosynthesis pathway reduces B cell growth, survival and proliferation

(A-D) B cells were purified from the spleens of C57BL6/J mice and cultured in normal or cholesterol-free (CF) media. The cells were stained with CTV, then pre-treated with DMSO as a vehicle control or Fluvastatin (10µM) or NB-598 (10µM), where indicated, for 45 minutes prior to stimulation with LPS (20μg/ml) and IL-4 (10μg/ml) for 48 hours. Gating strategy for CTV staining is shown in Figure S15B. (A) shows representative CTV staining, (B) live B cell number, (C) percentage of live B cells (7AAD-ve) and (D) forward scatter. Data shows the results of three biological replicates. (E-H) Same as (A-D) but with pretreatment using FGTI-2734 (10µM). (E) shows representative CTV staining, (F) live B cell number, (G) percentage of live B cells (7AAD-ve) and (H) forward scatter of B cells. Data shows the results of three biological replicates. (I-P) Same as (A-D) but with pretreatment using FTI-277 or GGTI-298 (10µM-30µM). (I) shows representative CTV staining in normal media, (J) live B cell number, (K) percentage of live B cells (7AAD-ve) and (L) forward scatter of B cells. (M) shows representative CTV staining in CF media, (N) live B cell number, (O) percentage of live B cells (7AAD-ve) and (P) forward scatter of B cells. Data shows the results of three biological replicates. (B-D) (F-H) Statistical power was determined using two-way ANOVA followed by multiple comparison testing via Tukey’s analysis. For comparisons to the LPS + IL-4 condition, p<0.01 is indicated by **, p<0.001 by *** and p<0.0001 by ****. ns indicated by p>0.05. (J-L) (N-P) Statistical power was determined using one-way ANOVA followed by multiple comparison testing via Dunnett’s analysis. For comparisons to the LPS + IL-4 condition, p<0.01 is indicated by **, p<0.001 by *** and p<0.0001 by ****. ns indicated by p>0.05. For all panels, cells in the absence of LPS + IL-4 were naïve B cells analysed on the day of isolation.

Mevalonate supplementation rescues the effect of Fluvastatin treatment in B cells

(A-B) Splenocytes from C57BL6/J mice were plated in normal or cholesterol-free (CF) media and pre-treated with HEPES as a vehicle control or mevalonate (2mM) for 1 hour prior to treatment with DMSO or Fluvastatin (10µM), where indicated, for 45 minutes before stimulation with LPS (20μg/ml) and IL-4 (10ng/ml). The cells were fixed after 24 hours or 48 hours of LPS + IL-4 stimulation and stained with filipin. (A) Filipin staining comparing B cells +/- Fluvastatin or mevalonate after 24 or 48 hours of LPS + IL-4 stimulation in normal media. (B) Filipin staining comparing B cells +/- Fluvastatin or mevalonate after 24 or 48 hours of LPS + IL-4 stimulation in CF media. Data shows the results of three biological replicates. (C-F) B cells were purified from the spleens of C57BL6/J mice and cultured in normal or cholesterol-free media. The cells were stained with CTV, then pre-treated with HEPES as a vehicle control or mevalonate (2mM) for 1 hour. The cells were treated with DMSO or Fluvastatin (10µM), where indicated, for 45 minutes prior to stimulation with LPS (20μg/ml) and IL-4 (10μg/ml) for 48 hours. (C) Shows percentage of live B cells (7AAD-ve), (D) forward scatter of B cells, (E) representative CTV staining and (F) live B cell number. Data shows the results of three biological replicates. Where shown, statistical power was determined using two-way ANOVA followed by multiple comparison testing via Sidak’s analysis. For comparison to Fluvastatin treated B cells, p<0.05 is indicated by *, p<0.01 by **, p<0.001 by ***, p<0.0001 by ****. ns by >0.05. For all panels, cells in the absence of LPS + IL-4 were naïve B cells analysed on the day of isolation.

GGPP supplementation rescues the effect of Fluvastatin treatment in B cells

(A-H) B cells were purified from the spleens of C57BL6/J mice and cultured in normal or cholesterol-free media. The cells were stained with CTV, pre-treated with methanol:ammonium hydroxide solution (CH3OH:NH4OH) as a vehicle control or geranylgeranyl pyrophosphate (GGPP) (10μM) for 1 hour before treatment with Fluvastatin (10μM), where indicated. The cells were stimulated with LPS (20μg/ml) and IL-4 (10μg/ml) and cultured for 48 hours. (A) shows representative CTV staining for B cells cultured in normal media, (B) live B cell number, (C) percentage of live B cells and (D) forward scatter of B cells. (E) shows representative CTV staining for B cells cultured in CF media, (F) live B cell number, (G) percentage of live B cells and (H) forward scatter of B cells. Data shows the results of three biological replicates. Statistical power was determined using one-way ANOVA followed by multiple comparison testing via Dunnett’s analysis. For comparison to Fluvastatin treated B cells, p<0.001 is indicated by *** and p<0.0001 by ****. ns indicated p>0.05. For (B) and (F) this data was log-transformed then statistically analysed due to unequal variance. (I-J) Splenocytes were plated in normal or cholesterol-free media and pre-treated with methanol:ammonium hydroxide solution (CH3OH:NH4OH) as a vehicle control or geranylgeranyl pyrophosphate (GGPP) (10μM), where indicated, for 1 hour before treatment with DMSO or Fluvastatin (10μM). The cells were then stimulated with LPS (20μg/ml) and IL-4 (10ng/ml). The cells were fixed after 24 hours and stained with filipin before acquisition. (I) Filipin staining comparing B cells +/- Fluvastatin or GGPP after 24 or 48 hours of LPS + IL-4 stimulation in normal media. (J) Filipin staining comparing B cells +/- Fluvastatin or GGPP after 24 or 48 hours of LPS + IL-4 stimulation in CF media. Data shows the results of three biological replicates. Statistical power was determined using two-way ANOVA followed by multiple comparison testing via Sidak’s analysis. For comparison to Fluvastatin treated B cells, p<0.01 is indicated by **, p<0.001 by *** and p<0.0001 by ****. ns indicated p>0.05. For all panels, cells in the absence of LPS + IL-4 were naïve B cells analysed on the day of isolation.

MAPK and mTOR signalling regulate B cell proliferation and cholesterol levels

(A-E) B cells were purified from the spleens of C57BL6/J mice and stained with CTV, then pre-treated with DMSO as a vehicle control, PD18352 (2μM), VX745 (1μM) or Rapamycin (20nM), where indicated, for 45 minutes prior to stimulation with LPS (20μg/ml) and IL-4 (10μg/ml) for 48 hours. (A) shows representative CTV staining, (B) percentage of B cells per generation quantified from (A), (C) live B cell number (D) percentage of live B cells (7AAD-ve) and (E) forward scatter. Data shows three technical replicates from cells isolated from one mouse and is representative of three independent experiments. Statistical power for (B) was determined using two-way ANOVA followed by multiple comparison testing via Sidak’s analysis. For comparison to LPS + IL-4 stimulated B cells, p<0.0001 by **** and ns by >0.05. (F) Splenocytes from C57BL6/J mice were pre-treated with DMSO as a vehicle control, PD18352 (2μM), VX745 (1μM) or Rapamycin (20nM), where indicated, for 45 minutes prior to stimulation with LPS (20μg/ml) and IL-4 (10μg/ml). The cells were fixed after 24 hours and stained with filipin before acquisition. (F) Filipin staining after inhibitor treatment. Statistical power was determined by one-way ANOVA followed by multiple comparison testing via Dunnett’s analysis, where p<0.05 is indicated by *, p<0.01 by **, p<0.001 by ***, p<0.0001 by **** and ns by >0.05. For all panels, cells in the absence of LPS + IL-4 were naïve B cells analysed on the day of isolation.

Cholesterol is required for the growth and proliferation of B cells by multiple stimuli

(A) Splenocytes from C57BL6/J mice were plated in normal or CF media, before stimulation with LPS (20μg/ml), IL-4 (10μg/ml) or a combination of LPS and IL-4. stimuli. The cells were fixed after 24 hours and stained with filipin. (A) Filipin staining comparing cholesterol content between different stimuli. Data shows the results of three biological replicates. Statistical power was determined using two-way ANOVA followed by multiple comparison testing via Tukey’s analysis. For comparisons to naïve B cells, p<0.0001 is indicated by ****. (B) Splenocytes from C57BL6/J mice were plated in normal media before stimulation with LPS (20μg/ml), Resiquimod (1μg/ml), ODN 1826 (1μg/ml), Anti-IgM (10μg/ml), or CD40L (500ng/ml). The cells were fixed after 24 hours and stained with filipin. (B) Filipin staining comparing cholesterol content between different stimuli. Data shows the results of three biological replicates. Statistical power was determined using one-way ANOVA followed by multiple comparison testing via Dunnett’s analysis. For comparisons to naïve B cells, p<0.0001 is indicated by ****. (C-E) B cells were purified from the spleens of C57BL6/J mice and cultured in normal media. The cells were stained with CTV, then pre-treated with DMSO as a vehicle control or Fluvastatin (10µM) for 45 minutes before stimulation with LPS (20μg/ml), Resiquimod (1μg/ml), ODN 1826 (1μg/ml), Anti-IgM (10μg/ml), or CD40L (500ng/ml) as indicated for 72 hours. (C) shows the percentage of live B cells (7AAD-ve), (D) representative histogram for CTV staining and (E) live B cell number. Data shows the results of three biological replicates. Statistical power was determined using two-way ANOVA followed by multiple comparison testing via Tukey’s analysis, where p<0.0001 is indicated by ****. For (E), this data was log-transformed and then statistically analysed due to unequal variance. Statistical power was determined using two-way ANOVA followed by multiple comparison testing via Sidak’s analysis, where p<0.0001 is indicated by ****.

MACS antibodies

Inhibitors

Flow cytometry antibodies

Western blot primary antibodies