Journal: bioRxiv

Article Title: Synthetic auxotrophy reveals metabolic regulation of plasma cell generation, affinity maturation, and cytokine receptor signaling

doi: 10.1101/2025.05.05.652119

Figure Lengend Snippet: (a) B cells were activated and cultured under conditions promoting plasma cell differentiation in the presence of the indicated combinations of vehicle (DMSO) or inhibitors of GLS (CD839) and the MPC (UK5099). Shown are representative histograms from flow cytometric analyses of CD138 within the live cell gate, with inset numbers denoting the %CD138 + . The bar graph shows the mean (±SEM) results for generation of CD138 + cells under each treatment condition, pooling data from three temporally independent experiments, each with 3-5 independent B cell pools purified from separate mice (each dot represents a distinct sample). (b) Shown are the mean (±SEM) calculated numbers of PC generated in temporally independent replica experiments with a total of eight independent B cell pools cultured in vitro in (a). (c) Representative ELISpot results measuring the frequencies of IgM- and IgG1-secreting PC, as indicated, produced in the differentiation cultures under each treatment condition. (d) Bar graphs with mean (±SEM) ELISpot results pooled from the replicate experiments illustrated in (c), with each dot representing an individual sample. Shown are data normalized to the vehicle (DMSO) control for each set of cultures using an individual B cell pool. (e) Bar graphs show the mean (±SEM) absorbance values from ELISA measurements of IgM and IgG1 secreted into the media during the cultures as in (b). Additional data quantifying ASCs are presented in . (f) Prdm1 gene expression promoted by GLS and MPC2. B cells were activated and cultured as above, but harvested after 3.5 d culture in BAFF, IL-4, IL-5, and the indicated inhibitor(s) or vehicle followed by qRT2-PCR to quantitate Prdm1 RNA encoding Blimp1. Shown are the results from four biologically independent mouse pools, B cell purifications and cultures, with the Prdm1 -encoded RNA then normalized in each experiment to the level in the vehicle (DMSO) control (in each sample, relative to the averaged C T values of cyclophilin A and GAPDH). (g, h) Global gene expression identifies plasma cell identity as a main target of synthetic auxotrophy. Using three biologically independent replicate pools for each condition, RNA-seq was performed with the B cells cultured as in (f). Enriched genes identified by DESeq2 comparison were analyzed using the MyGeneset tool from ImmGen. (g) Genes enriched in vehicle treated cultures compared to cultures treated with both CB839 and UK5099 are shown as a W-plot with defined stages for mature B cells and PC indicated. (h) Genes enriched in CB839-treated cultures compared to cultures treated with both CB839 and UK5099 are shown as a heatmap of z-scored relative expression, with specific gene identities and defined stages for mature B cells and PC indicated. (i) Metabolic mitigation of the block imposed by synthetic auxotrophy. Graphs of aggregate results from six biologically independent B cell preparations (two biological replicates in each of three independent experiments), presented as in (a), are shown for differentiation assays performed with B cells purified, activated, and cultured as in (a), except that the cell permeable αKG analogue DMK was added as indicated. (j-l) Gene set enrichment analyses were performed on RNA-seq data generated using RNA from flow-purified GC B cells and hallmark gene sets of the Mouse Molecular Signatures Database. Shown is a selected subset of analyses with high normalized enrichment scores (NES) for the indicated gene sets (j) oxidative phosphorylation (WT vs Gls Δ/Δ, Mpc2 Δ/Δ GC B cells); (k) regulated by c-Myc ( Mpc2 Δ/Δ vs Gls Δ/Δ, Mpc2 Δ/Δ GC B cells); (l) oxidative phosphorylation ( Mpc2 Δ/Δ vs Gls Δ/Δ, Mpc2 Δ/Δ GC B cells). Additional GSEA and other data are in ).

Article Snippet: To test how inhibitors affected in vitro proliferation, cytokine receptor signaling, or plasma cell differentiation, cultures were supplemented with 1 μM CB839, 10 μM UK5099, and/or 3 μM hydroxychloroquine (all from Cayman Chemical Co, Ann Arbor MI), individually or in combinations, and 1 mM DMK was used to supplement some cultures subjected to glutaminolysis inhibition by CB839.

Techniques: Cell Culture, Clinical Proteomics, Cell Differentiation, Purification, Generated, In Vitro, Enzyme-linked Immunospot, Produced, Control, Enzyme-linked Immunosorbent Assay, Gene Expression, RNA Sequencing, Comparison, Expressing, Blocking Assay, Phospho-proteomics

Journal: bioRxiv

Article Title: Synthetic auxotrophy reveals metabolic regulation of plasma cell generation, affinity maturation, and cytokine receptor signaling

doi: 10.1101/2025.05.05.652119

Figure Lengend Snippet: Using conditions akin to , purified B cells were activated and cultured 4 d in the presence or absence of the indicated inhibitors. Equal number of viable cells were replated for ELISpot assays after rinsing and counting, with cultures in the wells performed in the absence (panels a, c) or presence (b, d) of the indicated inhibitors. (a, b) Photographs of spots in wells of the indicated cultures, scoring the frequencies as well as spot sizes of cells secreting IgM or IgG1 as indicated. Shown are single wells from one representative experiment, representative of the technical duplicates and of the five biological replicate experiments. (a) B cells were cultured 4 d in the presence of CB839 or UK5099 prior to plating culturing in inhibitor-free medium for the ELISpots. (b) B cells were cultured 4 d in inhibitor-free medium, followed by addition of the indicated compounds after plating in the ELISpot wells and overnight cultures. (c, d) Mean (±SEM) data from all five biologically independent replicate experiments quantitating the relative frequencies of ASCs and sizes of the spots (surrogates for amount of Ab secreted during the overnight culture) are shown. Left, middle, and right panels show relative frequencies of ASCs secreting IgG1, mean spot sizes after detection of IgM, and mean IgG1 spot sizes, respectively. For each experiment (a common pool of purified B cells activated and cultured in parallel with inhibitor(s) or vehicle alone), the ASC numbers and average spot sizes for inhibitor-treated cultures were normalized to those measured for the vehicle (DMSO) control. (c) Inhibitors were present during 4 d cultures, as in (a). (d) Inhibitors were added only after plating in ELISpot wells for overnight assays of secretion after a pool of activated B cells was aliquoted after culture 4 d without inhibitor present,

Article Snippet: To test how inhibitors affected in vitro proliferation, cytokine receptor signaling, or plasma cell differentiation, cultures were supplemented with 1 μM CB839, 10 μM UK5099, and/or 3 μM hydroxychloroquine (all from Cayman Chemical Co, Ann Arbor MI), individually or in combinations, and 1 mM DMK was used to supplement some cultures subjected to glutaminolysis inhibition by CB839.

Techniques: Purification, Cell Culture, Enzyme-linked Immunospot, Control

Journal: bioRxiv

Article Title: Synthetic auxotrophy reveals metabolic regulation of plasma cell generation, affinity maturation, and cytokine receptor signaling

doi: 10.1101/2025.05.05.652119

Figure Lengend Snippet: (a) Pools of purified B cells from WT mice or those with the indicated gene-targeted loss(es) of function were activated and cultured (1 and 2 d) with αCD40, BAFF, IL-4, and IL5. Metabolic functions were then assayed by a metabolic flux analyzer. (b-f) As for (a), except WT cells were treated with inhibitors (CB839; UK5099) alone or in combination, as indicated by the color coding, and then subjected to mitochondrial stress-tested measurements of respiration (b), biochemical assays of [ATP] (g-i), flow cytometry (j, l) or qPCR (k). (a) Oxygen consumption rates (OCR) during mitochondrial stress testing of B cells, comparing loss-of-function B cells of the indicated genotypes, color-coded as in . (b-d) Changes in basal respiration and maximal respiration of B cells from day 1 to day 2 after activation (b), calculated from assays in (c) and (d) OCR values at day 2 were used for statistical analysis. (c, d) As in (a) except that purified WT B cells were used, with additions of vehicle (DMSO) or the indicated inhibitor(s) (CB839, 1 µM; UK5099, 10 µm), with each B cell pool assayed on both days 1 (c) and 2 (d) after purification and activation. (e) Extra-cellular acidification rates (ECAR) during glycolytic stress tests of WT B cells activated and cultured in the presence of the indicated agents after 2 d cultures as in (d). (f) ECAR during glycolytic stress test of B cells inducibly rendered Gls Δ/Δ and/or Mpc2 Δ/Δ, then activated and cultured as in (a). (g, h, i) Intracellular [ATP] in lysates of B cells activated and cultured (2 d) as in (c). (g) Metabolic inhibitor(s) were present throughout the period of culture (2 d) and assay. (h) Cells were activated and initially cultured in the presence of the indicated metabolic inhibitor(s), then washed, and assayed (90 min) in medium without inhibitors. (i) After activation and 2 d culture with no inhibitor present, the indicated agent(s) were added to block glutaminolysis and/or mitochondrial pyruvate import during the 90-minute assay. (j) Mitochondrial membrane potential determined by tetramethylrhodamine (TMRE) staining analyzed by flow cytometry. Shown are mean fluorescence intensity (MFI) values from each independent experiment after activation and culture (2 d) as in (c), then normalized to DMSO-treated condition in each experiment. (k) Flow cytometry results comparing inhibitor-treated cells vs controls after staining for ROS with DCFDA in three independent replication experiments, with each dot representing one experiment, normalizing as in (i).

Article Snippet: To test how inhibitors affected in vitro proliferation, cytokine receptor signaling, or plasma cell differentiation, cultures were supplemented with 1 μM CB839, 10 μM UK5099, and/or 3 μM hydroxychloroquine (all from Cayman Chemical Co, Ann Arbor MI), individually or in combinations, and 1 mM DMK was used to supplement some cultures subjected to glutaminolysis inhibition by CB839.

Techniques: Purification, Cell Culture, Flow Cytometry, Activation Assay, Blocking Assay, Membrane, Staining, Fluorescence

Journal: bioRxiv

Article Title: Synthetic auxotrophy reveals metabolic regulation of plasma cell generation, affinity maturation, and cytokine receptor signaling

doi: 10.1101/2025.05.05.652119

Figure Lengend Snippet: (a) Enrichment of the IL6-stimulated Jak-STAT3-induced gene set. (b) Immunoblot analyses of IL-21-induced tyrosine phosphorylation of STAT3 in B cell blasts, showing representative results from one individual experiment representative of three independent replications. Purified B cells were activated with anti-CD40 and BAFF, cultured for 16 hr in the presence of vehicle (DMSO) or inhibitors (CB839 and UK5099), as indicated, then stimulated (15 min) with IL-21. (c) Results from an over-representation analysis of differentially expressed protein-coding RNA in WT versus induced double-deficient (GLS; MPC2; "diKO B ") B cells, as counted in the RNA-seq analyses with GC B cells of SRBC-immunized mice (as in ). Additional GSEA and an overview of gene set comparisons for pairs of genotypes are presented in . (d) Selected analyses of gene sets enriched in WT GCBs compared to diKOi B GCBs using the Hallmark Pathway database. Shown are enrichment of IFN-α- and IFN-γ-associated pathways in WT GCBs compared to the Gls Δ/Δ, Mpc2 Δ/Δ samples. ( e-j ) Immunoblot analyses of IFN-induced STAT1 phosphorylation in activated B cells, showing representative results from individual experiments, each representative of three independent replications. (e, f) Purified B cells were activated with anti-CD40 and BAFF and cultured for 64 or 16 hr in the presence of vehicle (DMSO) or inhibitors (CB839 and UK5099), as indicated, then stimulated (15 min) with IFN-β (e) or IFN-γ (f) followed by immunoblotting using Ab specific for p-STAT1(Y701) or p-STAT1α(S727) along with anti-cyclophilin B Ab as a loading control. ( g, h ) Inhibition of mitochondrial ETC attenuates STAT1 activation. B cells were activated and cultured as in e and f but for 16 hr, in the presence or absence of metformin (2 mM; 16 hr) or rotenone (0.5 µM; final 2 h of the cultures) as indicated, then stimulated (15 min) with IFN-β (g) or IFN-γ (h) and analyzed as for panels d, e. (i, j) ROS inhibit STAT1 activation. B cells were activated and cultured for 16 hr, in the presence or absence of menadione (2 µM; all 16 h) or H 2 O 2 (100 µM; the final 2 h), and then stimulated with IFN-β (i) or IFN-γ (j) for 15 min.

Article Snippet: To test how inhibitors affected in vitro proliferation, cytokine receptor signaling, or plasma cell differentiation, cultures were supplemented with 1 μM CB839, 10 μM UK5099, and/or 3 μM hydroxychloroquine (all from Cayman Chemical Co, Ann Arbor MI), individually or in combinations, and 1 mM DMK was used to supplement some cultures subjected to glutaminolysis inhibition by CB839.

Techniques: Western Blot, Phospho-proteomics, Purification, Cell Culture, RNA Sequencing, Control, Inhibition, Activation Assay