SREBP signaling is essential for effective B cell responses

Abstract

Our previous study using systems vaccinology identified an association between the sterol regulatory binding protein (SREBP) pathway and humoral immune response to vaccination in humans. To investigate the role of SREBP signaling in modulating immune responses, we generated mice with B cell- or CD11c⁺ antigen-presenting cell (APC)-specific deletion of SCAP, an essential regulator of SREBP signaling. Ablation of SCAP in CD11c⁺ APCs had no effect on immune responses. In contrast, SREBP signaling in B cells was critical for antibody responses, as well as the generation of germinal centers,memory B cells and bone marrow plasma cells. SREBP signaling was required for metabolic reprogramming in activated B cells. Upon mitogen stimulation, SCAP-deficient B cells could not proliferate and had decreased lipid rafts. Deletion of SCAP in germinal center B cells using AID-Cre decreased lipid raft content and cell cycle progression. These studies provide mechanistic insights coupling sterol metabolism with the quality and longevity of humoral immunity.

Extended Data Fig. 1. SREBP signaling in CD11c+ antigen presenting cells is dispensable for modulation of antibody and CD8 T cell responses.

a. Spleen single cell suspension from SCAP^fl/fl (n = 4) and SCAP^fl/fl CD11c-Cre mice (n = 6) were analyzed by flow cytometry for dendritic cell (DC) subtypes. DC subtypes are gated as cDCs (CD45⁺CD11c⁺MHCII⁺) which include cDC1 (CD8a⁺CD11b⁻) and cDC2 (CD8a⁻CD11b⁺), pDCs (CD45⁺CD11c^loPDCA1⁺). Graphs show the representative flow cytometry plots and the statistics for the frequencies of DC subsets. b. SCAP^fl/fl and SCAP^fl/fl CD11c-Cre mice were immunized subcutaneously with OVA adjuvanted with RIBI (n = 12 for SCAP^fl/fl and n = 9 for SCAP^fl/fl CD11c-Cre) or AddaVax (n = 8 for each group), and serum antibody titers at day 28 were measured by ELISA. c. SCAP^fl/fl and SCAP^fl/fl CD11c-Cre mice (n = 9 for each group) were immunized with 2×10⁶ plaque-forming units (PFU) of YF-17D virus, and antigen specific CD8 T cells producing IFN-γ in the lung and liver were analyzed by flow cytometry. Graphs show frequencies of CD8 T cell (gated as CD3⁺CD8⁺CD4⁻) producing IFN-γ in the lung and liver with representative flow cytometry plots. a-c, Data represent two independent experiments, P values are determined by two-tailed unpaired t-test. ns: P > 0.05.

Extended Data Fig. 2. SCAP is not required for B cell development or maintenance at steady state.

Splenic B cell subsets from SCAP^+/+CD19^Cre/+ (+/+) mice and SCAP^fl/fl CD19^Cre/+ (fl/fl) mice were analyzed by FACS. a. Gating strategy. b. Representative FACS plots comparing total B cells, and the follicular and marginal zone B cell subsets. c. Statistical analysis of B cell subsets at steady state. b and c, data are from 2 experiments (n = 6, mean ± SD). P values were determined by two-tailed unpaired t-test. ns, P > 0.05. d. Lipid raft staining by cholera toxin subunit B (CT-B) of purified B cells. Shown are representative images of 4 samples from 2 experiments. e. FACS analysis of purified B cells stimulated with 20 μg/ml anti-IgM antibody for indicated time points. Data represent 5 mice from 2 experiments. ns: P > 0.05.

Extended Data Fig. 3. SREBP signaling is required for MBC formation.

Mice were i.p immunized with NP-OVA adjuvanted by RIBI. Splenocytes were analyzed 4 weeks post immunization. a. Gating strategy to identify isotype switched MBC (B220⁺CD95⁻CD38⁺IgM⁻IgD⁻) and subsets of these MBC based on CD80 and PD-L2 expression. b. Dot plots with means are from 2 independent experiments (n = 8 for SCAP^+/+ CD19^Cre/+ and n = 8 for SCAP^fl/fl CD19^Cre/+). P values were determined by two-tailed unpaired t-test. ***P ≤ 0.001, ****P ≤ 0.0001.

Extended Data Fig. 4. SREBP signaling does not affect early IgM antibody response.

wk1 and wk2 serum samples from Fig. 1a–c were analyzed by ELISA for NP binding IgM antibody titers (n = 11 from two independent experiments). Shown are dot plots with means. P values were determined by two-tailed unpaired t-test. ns: P > 0.05.

Extended Data Fig. 5. Steroid biosynthesis (including terpenoid backbone biosynthesis) KEGG pathway annotated with gene expression.

Bead-purified splenic B cells from SCAP^+/+ CD19^Cre/+ (+/+) mice and SCAP^fl/fl CD19^Cre/+ (fl/fl) mice were stimulated with 5 μg/ml anti-CD40 tetramer for 24 hours. RNA was isolated from the cells and analyzed by RNA sequencing.

Extended Data Fig. 6. RNA-seq analysis reveals altered pathways in LPS or anti-IgM stimulated SCAP deficient B cells.

Bead-purified splenic B cells from SCAP^+/+ CD19^Cre/+ mice and SCAP^fl/fl CD19^Cre/+ mice were stimulated with 10 μg/ml LPS or 20 μg/ml anti-IgM for 24 hours. RNA was isolated from the cells and analyzed by RNA sequencing. Pathways that were altered in SCAP deficient B cells post LPS (a) and anti-IgM (b) stimulation were ranked by FDR ( < 0.05). Shown are data from 2 independent experiments with cells pooled from 5 mice in each experiment.

Extended Data Fig. 7. SCAP deficiency leads to reduced survival of B cells activated by BCR signal.

B cells isolated from SCAP^+/+ CD19^Cre/+ (+/+) mice and SCAP^fl/fl CD19^Cre/+ (fl/fl) mice were stimulated with 20 μg/ml anti-IgM for 2 days. Cell viability was measured by a cell counter with AO/PI staining. Data represent 7 biological replicates from 2 independent experiments. P value is determined by two-tailed unpaired t-test. ****P ≤ 0.0001.

Extended Data Fig. 8. Altered signaling in activated SCAP deficient B cells.

B cells isolated from SCAP^+/+CD19^Cre/+ mice and SCAP^fl/flCD19^Cre/+ mice were stimulated with 10 μg/ml LPS, 5 μg/ml anti-CD40 or 20 μg/ml anti-IgM for 0, 4 and 24 hours. Cell lysates were analyzed by immunoblotting. a. Representative immunoblotting. b. Quantitation of 2 independent immunoblotting experiments. c. B cells isolated from SCAP^+/+ CD19^Cre/+ mice and SCAP^fl/fl CD19^Cre/+ mice were stimulated with 10 μg/ml LPS and cultured with or without 5 μg/mL MβCD conjugated cholesterol for 24 hours. Data shown is a representative immunoblotting of 2 independent experiments.

Extended Data Fig. 9. Metabolomics reveals global metabolic changes in SCAP deficient B cells.

Splenic B cells isolated from SCAP^+/+ CD19^Cre/+ (+/+) mice and SCAP^fl/fl CD19^Cre/+ (fl/fl) mice were stimulated with 10 μg/ml LPS or 5 μg/ml anti-CD40 tetramer for 24 and 48 hours. Cells were then analyzed by metabolomics. a. PCA plot showing the differences of all replicates between different treatments in different genotypes. b-c. Heatmap of altered metabolites (FDR < 0.05, log2 Fold Change >1) marked by their pathways. Colors represent log2 FC relative to the untreated corresponding genotype samples. Four biological replicates generated from two experiments were used for metabolomics analysis.

Extended Data Fig. 10. SREBP signaling regulates GC B cell response.

a-b. AID-Cre R26^YFP mice were immunized with NP-OVA adjuvanted by RIBI through i.p or s.c. Splenocytes (i.p immunization) and dLN cells (s.c immunization) were analyzed by FACS 10 days post immunization. GC B cells and CD86^hi MHCII^hi non-GC B cells were compared for Cre activity through their YFP reporter expression. a shows the gating strategy; b shows the statistical analysis of 2 independent experiments (n = 5, mean ± SD). c. SCAP^+/+ AID-Cre R26^YFP mice (+/+) and SCAP^fl/fl AID-Cre R26^YFP mice (fl/fl) were i.p. immunized with NP-OVA adjuvanted with RIBI. Cells in the spleen were analyzed by FACS 14 days post immunization for the % of YFP⁺ GC B cells in total GC B cells (gated on CD19⁺CD95⁺CD38⁻ live singlets). 10 + /+ and 14 fl/fl mice from 4 independent experiments were analyzed. a-c, P values were determined by two-tailed unpaired t-test. ****P ≤ 0.0001. d. Splenocytes from day 14 RIBI adjuvanted NP-OVA immunization were analyzed by FACS to compared lipid raft content between GC and non-GC B cells. Lipid rafts were stained with cholera toxin subunit B (CT-B). Data show one representative of two independent experiments.

Fig.1 |. B cell SREBP signaling is important for humoral immune response and memory formation.

a. Timeline of NP-OVA immunization and analysis. b. ELISA analysis for the kinetics of NP2- and NP14-binding antibody titers (mean ± SEM). c. Antibody forming plasma cells in the bone marrow were analyzed by ELISPOT assay. Data shown in b and c are comparing SCAP^fl/fl CD19^Cre/+ mice (n=11) with SCAP^+/+ CD19^Cre/+ (n=11) and SCAP^fl/fl CD19^+/+ (n=10) mice from 2 independent experiments. d. Mice were subcutaneously immunized with NP-OVA adjuvanted with RIBI. GC B cells and TFH in the draining lymph node were analyzed by FACS day 12 post immunization. Left panel shows the representative FACS plots; right panel shows the statistical analysis of 10 mice from 2 independent experiments. e. Timeline of YF-17D infection and analysis. f. ELISA analysis for the YF-17D binding antibody titer 2 and 6 weeks post YF-17D infection. Shown are data from 2 independent experiments comparing SCAP^fl/fl CD19^Cre/+ mice (n=5) with control mice (n=8). Control are SCAP^+/+ CD19^Cre/+ and SCAP^fl/fl CD19^+/+ mice. d and f, lines in dot plots are means. P values were determined by two-tailed unpaired t-test (b, d, f) or by one-way ANOVA followed by Tukey’s multiple comparisons test (c). P values are *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001, ns: P>0.05. (blue stars are SCAP^+/+ CD19^Cre/+ versus SCAP^fl/fl CD19^Cre/+ and green stars are SCAP^fl/fl CD19^+/+ versus SCAP^fl/fl CD19^Cre/+).

Fig.2 |. RNA-seq analysis for pathways associated with SREBP signaling in B cells.

a-b. Splenic B cells were bead-purified from SCAP^+/+ CD19^Cre/+ (+/+) mice and SCAP^fl/fl CD19^Cre/+ (fl/fl) mice. Cells were then stimulated with 10μg/ml LPS, 5μg/ml ⍺-CD40 tetramer or 20μg/ml ⍺-IgM for 0 (as control) and 24 hours. RNA was isolated from the cells and analyzed by RNA sequencing. a. Heat map shows the change of genes involved in sterol biosynthesis and fatty acid metabolism. b. Pathways ranked by FDR (<0.05) that were altered in SCAP^fl/fl CD19^Cre/+ mice compared to SCAP^+/+ CD19^Cre/+ mice 24 hours post ⍺-CD40 stimulation. RNA samples were from 2 independent experiments with cells pooled from 5 mice in each experiment.

Fig.3 |. B cell SREBP signaling is required for mitogen stimulated cell cycle progression.

a-b. Splenic B cells isolated from SCAP^+/+ CD19^Cre/+ (+/+) mice and SCAP^fl/fl CD19^Cre/+ (fl/fl) mice were labeled with CFSE. Then cells were stimulated with 10μg/ml LPS, 10μg/ml CpG or 10μg/ml R848 for 3 days, or 5μg/ml ⍺-CD40 tetramer for 5 days. Cell proliferation was examined by CFSE dilution using FACS. a. The representative histogram of CFSE dilution; b. The statistical analysis of 5 mice from 2 independent experiments (mean ± SD). c-d. Splenic B cells were stimulated with 10μg/ml LPS, 5μg/ml ⍺-CD40 tetramer or 20μg/ml ⍺-IgM for 48 hours. Cell cycle phases (G1, S, G2/M) were analyzed by FACS with EdU and DAPI. c. The representative FACS plots showing EdU and DAPI staining. d. The statistical analysis of 4 mice from 2 independent experiments (mean). e-f. Splenic B cells were stimulated with 10μg/mL LPS and cultured with or without 5μg/mL MβCD conjugated cholesterol for 3 days. e. The representative histogram of CFSE dye dilution. f. Quantification of B cell division of 4 +/+ and 3 fl/fl mice from 2 independent experiments (mean ± SD). g-h. Splenic B cells were stimulated with 10μg/mL LPS or 5μg/ml α-CD40 tetramer and cultured with or without 5μg/mL MβCD conjugated cholesterol for 48 hours. Lipid raft was analyzed by FACS with CT-B staining. g. The histogram of CT-B staining. h. The statistical analysis of 5 mice from 2 independent experiments (mean ± SD). MFI values were normalized to +/+ samples with control given a value of 1. P values were determined by two-tailed unpaired t-test (b, d) or by one-way ANOVA followed by Tukey’s multiple comparisons test (f, h). *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001, ns: P>0.05.

Fig.4 |. SREBP signaling regulates energy metabolism in activated B cells.

a-c. B cells isolated from SCAP^+/+ CD19^Cre/+ (+/+) mice and SCAP^fl/fl CD19^Cre/+ (fl/fl) mice were stimulated with 10μg/ml LPS, 5μg/ml ⍺-CD40 tetramer or 20μg/ml ⍺-IgM for 48 hours. Seahorse technology was used to examine cell energy metabolism. Mitochondrial oxidative phosphorylation and glycolysis were evaluated by oxygen consumption rate (OCR) and proton efflux rate (PER), respectively. a. The representative data of OCR (n=5 for LPS and ⍺-CD40; n=4 for ⍺-IgM) and PER (n=5 for all treatments). Data represents 2 independent experiments. b-c. The statistical analysis. Data are mean ± SEM. P values were determined by two-tailed unpaired t-test. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001, ns: P>0.05.

Fig.5 |. Metabolomics reveals global metabolic changes in SCAP deficient B cell.

a-c. Splenic B cells isolated from SCAP^+/+ CD19^Cre/+ (+/+) and SCAP^fl/fl CD19^Cre/+ (fl/fl) mice were stimulated with 10μg/ml LPS or 5μg/ml α-CD40 tetramer for 24 and 48 hours. Cells were then analyzed by metabolomics. a. PCA plot showing the differences of all replicates between different treatments in different genotypes of unstimulated and 48 hours stimulated samples. b. Venn diagram of unique and common metabolites significantly differentially altered (FDR < 0.05, log2 Fold Change >1) in SCAP deficient B cells compared to WT B cells upon 48 hours stimulation of LPS and ⍺-CD40. c. Heatmap of altered metabolites (FDR < 0.01, log2 Fold Change >1.5) marked by their pathways. Colors represent log2 FC relative to the untreated corresponding genotype samples. Four biological replicates generated from two experiments were used for metabolomics analysis.

Fig.6 |. Control of GC B cell functions by SREBP signaling.

a. Splenic naïve B cells and GC B cells were isolated from naïve mice and day 8 SRBC i.p immunized mice, respectively. Immunoblotting was used for comparing protein expression between naïve B cells and GC B cells. Two experiments were performed with cells combined from 3 and 5 mice in each experiment. Right panel shows the densitometry analysis. Loadings are normalized to ACTIN and ratio of GC/naïve are shown as fold change. b. SCAP^+/+ AID-Cre R26^YFP (+/+) and SCAP^fl/fl AID-Cre R26^YFP (fl/fl) mice were i.p immunized with NP-OVA adjuvanted with RIBI. GC B cells in the spleen were analyzed by FACS 14 days post immunization. c. YFP⁺ splenic GC B cells generated in b were sorted and analyzed by RNA sequencing. Shown are top pathways (ranked by FDR) that were altered in SCAP^fl/fl AID-Cre R26^YFP GC B cells. d. FACS analysis of Cholera Toxin Subunit B (CT-B) labeling of GC B cells. b and d. 10 +/+ and 14 fl/fl mice from 4 independent experiments were analyzed. e. Day 14 NP-OVA immunized mice were i.v. injected with EdU. One hour post EdU labeling, mice were sacrificed, and GC B cells were analyzed for cell cycle by FACS. Statistical analysis of % of S phase represents 7 +/+ and 11 fl/fl mice from 3 independent experiments. f. Splenic cells from b were analyzed for CD138⁺ YFP⁺ plasma cells. Data represent 10 +/+ and 14 fl/fl mice from 4 independent experiments. g. SCAP^+/+ AID-Cre R26^YFP (+/+) and SCAP^fl/fl AID-Cre R26^YFP (fl/fl) mice were s.c immunized with RIBI adjuvanted NP-OVA. NP2 and NP14 binding antibody titers and their ratio were analyzed by ELISA. Data represent 7 +/+ and 11 fl/fl mice from 2 independent experiments. P values were determined by two-tailed unpaired t-test. **P≤0.01, ***P≤0.001, ****P≤0.0001.