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.
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Dynamic mitochondrial transcription and translation in B cells control germinal center entry and lymphomagenesis
Nature Immunology Open Access 24 April 2023
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RNA-seq data generated in this study have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus database under accession codes GSE204913 and GSE206016. Source data are provided with this paper.
All analyses and visualizations were performed in R. Computer code is available upon reasonable request.
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We are grateful for the next-generation sequencing services provided by the Yerkes NHP Genomics Core, which is supported in part by NIH P51 OD011132. Sequencing data were acquired on an Illumina NovaSeq 6000 system, funded by NIH S10 OD026799. We thank R. Casellas (NIH) for providing the AID-Cre R26YFP mice. We acknowledge the NIH (grants R37 DK057665, R37 AI048638, U19 AI090023 and U19 AI057266), Bill and Melinda Gates Foundation and Soffer Endowment Fund and Open Philanthropy (to B.P.) for supporting this work in B.P.’s laboratory.
The authors declare no competing interests.
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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 SCAPfl/fl (n = 4) and SCAPfl/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+CD11cloPDCA1+). Graphs show the representative flow cytometry plots and the statistics for the frequencies of DC subsets. b. SCAPfl/fl and SCAPfl/fl CD11c-Cre mice were immunized subcutaneously with OVA adjuvanted with RIBI (n = 12 for SCAPfl/fl and n = 9 for SCAPfl/fl CD11c-Cre) or AddaVax (n = 8 for each group), and serum antibody titers at day 28 were measured by ELISA. c. SCAPfl/fl and SCAPfl/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+/+CD19Cre/+ (+/+) mice and SCAPfl/fl CD19Cre/+ (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+/+ CD19Cre/+ and n = 8 for SCAPfl/fl CD19Cre/+). 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+/+ CD19Cre/+ (+/+) mice and SCAPfl/fl CD19Cre/+ (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+/+ CD19Cre/+ mice and SCAPfl/fl CD19Cre/+ 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+/+ CD19Cre/+ (+/+) mice and SCAPfl/fl CD19Cre/+ (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+/+CD19Cre/+ mice and SCAPfl/flCD19Cre/+ 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+/+ CD19Cre/+ mice and SCAPfl/fl CD19Cre/+ 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+/+ CD19Cre/+ (+/+) mice and SCAPfl/fl CD19Cre/+ (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 R26YFP 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 CD86hi MHCIIhi 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 R26YFP mice (+/+) and SCAPfl/fl AID-Cre R26YFP 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.
Source Data Fig. 5
Source data for metabolomics analysis.
Source Data Fig. 6
Unprocessed western blots for Fig. 6a.
Source Data Extended Data Fig. 8
Unprocessed western blots for Extended Data Fig. 8.
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Luo, W., Adamska, J.Z., Li, C. et al. SREBP signaling is essential for effective B cell responses. Nat Immunol 24, 337–348 (2023). https://doi.org/10.1038/s41590-022-01376-y