Glutamine and glutaminolysis promote antibody response to ovalbumin.

(a) Reduced glutamine concentration in LN relative to the bloodstream. Shown are the results from measuring concentrations glutamine, leucine, and pyruvate in the centrifugal eluates (67, 68) of lymph nodes or plasma of mice (n=3), as described in the Methods. (b) Mouse B cells were activated and cultured together BAFF, LPS, IL4, and IL5 in the presence of the indicated concentrations of Gln. Shown are the mean (±SEM) frequencies of PC (CD138+ B220lo; left panel) and IgG1+ B cells (right panel) after culture (4 d) and flow cytometry, averaging ≥ 3 independent replications. Results of measuring IgM and IgG1 by ELISA are shown in Supplemental Fig. 1a. (c) Reduction of PC differentiation and class switching caused by Gln restriction manifested at equal division numbers, and mitigated by a cell-permeable analogue of α-ketoglutarate. After labeling with CellTrace Violet, B cells were activated and cultured as in (b), except that dimethyl-ketoglutarate (DMK) was added to one of two cultures at 0.1 mM glutamine, as indicated. Shown are mean (±SEM) frequencies of % CD138+ (left panel) and IgG1+ B cells (right panel) within each division-counted peak. Additional data from these experiments are presented in Supplemental Fig 1b, c. (d-i) An anti-ovalbumin Ab response is promoted by B cell expression of GLS. (d) Schematic of the immunization with priming and sensitization of tamoxifen-treated mice of the indicated genotypes [huCD20-CreERT2+ and either Gls +/+ (WT) or Gls f/f], followed at week three by challenges with intranasal instillations of sterile ovalbumin solution. (e) Mediastinal LN were collected from harvested mice and the frequencies of IgG1+ events in the GL7+ CD95+ B cell gate were measured by flow cytometry. Each dot represents an individual mouse, with bars denoting the mean values. (f-h) Single-cell suspensions of lung and bone marrow were analyzed by ELISpot assays with ovalbumin-coated filters to capture secreted Ag-specific Ab detected using anti-mouse IgG1. Shown are (f) representative wells from the indicated sources (organ; genotype of B cells) and aggregated frequencies of anti-ova IgG1+ ASCs in lung (g) and marrow (h). (i) Anti-ova IgG1 in sera of the mice with B cell-restricted depletion of GLS (Gls iB-Δ/Δ) or controls, as indicated. Shown are data from ELISA with serial 4-fold dilutions using individual samples from each mouse. The likelihood of each null hypothesis (no true difference between two samples) was calculated as noted in the Methods (two-tailed, and non-parametric testing where conditions not met for Student’s t-test). *p<0.05, **p<0.01, *** p,0.001, ****p<0.0001.

Glutaminolysis only conditionally supports the anti-NP Ab response.

(a) Schematic of the immunization with priming sensitization and inhaled challenge of tamoxifen-treated mice of the indicated genotypes [huCD20-CreERT2+ and the indicated combinations of either Gls +/+ (WT) or Gls f/f and either Slc2a1 +/+ (WT) or Slc2a1 f/f], followed at week three by challenges with intranasal instillations of sterile ovalbumin solution. (b-f) ELISpot analyses of cells secreting IgG1 anti-ovalbumin (b, c) and anti-NP (d-f) Ab in mice of the indicated genotypes. Shown are counts from the lung suspensions (b), bone marrow (c, d, f) and spleens (d, e) from each individual mouse, with means (±SEMs) for the aggregate data denoted as bar graphs. (d) Representative ELISpot wells scoring the frequencies of IgG1 anti-NP Ab-secreting cells in spleen and marrow of mice whose B cells were converted to the indicated genotypes [wildtype (), GlsΔ/Δ (), Slc2a1Δ/Δ (), and GlsΔ/ΔSlc2a1Δ/Δ ()] after immunization and rechallenge. Data pooled from two independent experiments (seven wildtype, six GlsΔ/Δ, four Slc2a1Δ/Δ, and six GlsΔ/ΔSlc2a1Δ/Δ mice. * p < 0.05, ** p < 0.01, *** p < 0.001 (Mann-Whitney U test).(g-i). Shown are the mean (±SEM) absorbances of aggregated results of ELISA analyses of the IgM (g) and IgG1 (h, i) responses in the immunized mice of the two independent experiments, comparing all-affinity (h) to high-affinity (i) anti-NP, using serial 3-fold or 4-fold dilutions when detecting IgM and IgG1, respectively.

Synthetic auxotrophy - glutaminase support of anti-NP response is dependent on mitochondrial pyruvate channel subunit 2.

(a) Schematic of the immunization with priming and a recall boost of mice of the indicated genotypes [huCD20-CreERT2+ and the indicated combinations of either Gls +/+ (WT) or Gls f/f and either Mpc2 +/+ (WT) or Mpc2 f/f], treated with tamoxifen prior to the initial immunization. (b-d) Results of serological analyses performed on sera from the time of harvest. Shown are the mean (±SEM) absorbances measured in ELISA for detection of all- and high-affinity Abs (α-NP20 and α-NP2, respectively) of the (b) IgG1 and (c) IgG2c isotypes. Serologic measurements of IgM anti-NP Ab are shown in Supplemental Data Fig. 2a. (d) Impacts of altered metabolic pathways in B cells on relative extents of affinity maturation in the recall responses. Ratios of absorbances are shown for high- (NP2) / all-affinity (NP20) ELISA at two serum dilutions for the IgM, IgG1, and IgG2c isotypes, as indicated. (e-g) Shown are mean (±SEM) frequencies of splenocytes producing anti-NP Ab of the indicated isotypes [(e) IgM, (f) IgG1, (g) IgG2c] and affinities at harvest after the booster immunization, as in (a), with genotypes as shown and each dot representing an individual mouse. (b-g) Results are aggregated from four temporally separate immunization experiments with mice of each genotype, totaling eight individual controls [(WT, i.e., only CreERT2+) ()] and eight mice with each type of induced B cell type-specific deletion [GlsΔ/Δ (), Mpc2Δ/Δ (), and GlsΔ/Δ, Mpc2Δ/Δ () genotypes]. Supplemental Data Fig. 2 shows additional results, with frequencies of GC B cells, MBCs, BrdU uptake rates in GC B cells, as well as Gls and Mpc2 mRNA. (h, i) Functional requirements for GLS and MPC2 in B cells during the recall phase. (h) Schematic of the priming immunization without deletion of the conditional alleles, followed by tamoxifen injections ∼ 3 wk later, and only then a recall boost of mice of the indicated genotypes [huCD20-CreERT2+ and the indicated combinations of either Gls +/+ (WT) or Gls f/f and either Mpc2 +/+ (WT) or Mpc2 f/f]. (i) Shown are serologies of the all- and high-affinity IgG1 anti-NP Ab elicited by a boost when Cre activation was initiated only after the primary response. Results are aggregated from three temporally separate immunization experiments with mice of each genotype, totaling nine individual controls [(WT, i.e., only CreERT2+) ()] and nine mice with each type of induced B cell-specific deletion [GlsΔ/Δ (), Mpc2Δ/Δ (), and GlsΔ/Δ, Mpc2Δ/Δ () genotypes]. Supplemental Data Fig. 3 shows additional results, with IgM, IgG2c and IgA isotypes; frequencies of ASCs and NP-binding GC B cells.

GLS and MPC2 collaborate in supporting progression to plasma cell development.

(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 Supplemental Fig. 4. (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 CT 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 Supplemental Figure 7).

Synthetic auxotrophy of B cell metabolism that supports a progressive post-activation increase in mitochondrial respiration.

(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 Fig. 3. (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).

Metabolism in B cells promotes interferon receptor signaling to STAT1.

(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; "diKOB") B cells, as counted in the RNA-seq analyses with GC B cells of SRBC-immunized mice (as in Fig 4, j-l). Additional GSEA and an overview of gene set comparisons for pairs of genotypes are presented in Supplemental Fig. 6. (d) Selected analyses of gene sets enriched in WT GCBs compared to diKOiB 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 H2O2 (100 µM; the final 2 h), and then stimulated with IFN-β (i) or IFN-γ (j) for 15 min.

Glutamine and glutaminolysis promote an antibody response to ovalbumin.

Additional data from or relating to experiments in Figure 1. (a) Using a dilution in the linear range for the samples, relative concentrations of IgM (left panel) and IgG1 (right panel) in the supernatants of cultures of Fig. 1b were measured by ELISA, using titrations of re-added glutamine concentrations as indicated. Shown are the mean (±SEM) values from analysis of supernatants in three biologically independent experiments, with indications of P values as in the main figure. (b) Distributions of division counts under conditions of lower extracellular glutamine in experiments of Fig. 1b. Shown are the percentages of live cells in the gate for each peak of 2-fold CTV partitioning when grown under the indicated conditions. (c) Distributions of division counts under conditions of lower extracellular glutamine, with or without addition of dimethylketoglutarate (DMK) as indicated, in experiments of Fig. 1c. (d) Relative levels of ovalbumin-specific IgM in experiments of Fig. 1d-i, showing mean (±SEM) absorbances in ELISA data across serial dilutions, as in Fig. 1i.

Synthetic auxotrophy of glutaminolysis in primary anti-NP Ab responses.

(a-d) Mice of indicated genotypes were treated with tamoxifen and immunized as in Figure 3a. (a) All- and high-affinity (lower and upper, respectively) IgM anti-NP Ab in sera at time of harvest (same samples & display as in Fig 3b, c). (b-d) Spleens were harvested from NP-OVA boosted mice and the frequencies of GL7+ CD95+ GCB cells and GL7- CD38+ memory B cells (MBCs) were measured by flow cytometry. (b) Gating for measurements of prevalence in flow cytometric data, showing representative flow plots for GCB cells and MBCs in IgD- B cell gated cells. (c, d) Shown are mean (±SEM) frequencies of (c) GCB cells and (d) MBCs from five independent replicate experiments. (e) Intravital incorporation of BrdU by GCB cells after priming immunization, tamoxifen injection, and boost immunization as described in the Methods. (f, g) RNA-Seq normalized read counts of inducible gene knockout targets in flow sorted SRBC stimulated GCB cells. Related information is presented in Supplemental Fig. 8a, b.

Glutaminase and mitochondrial pyruvate channel promote response of reactivated memory B cells.

Mice of indicated genotypes were immunized with NP-OVA, injected with tamoxifen and boosted with NP-OVA as in Fig 3h. (a-c) Shown are serologies of the all- and high-affinity NP-specific IgM (a), IgG2c (b) and IgA (c) antibodies from 3 independent experiments. (d-f) Shown are mean (±SEM) numbers of NP-specific IgM-, IgG1-, and IgA-secreting cells in the splenocytes from immunized mice. (g-i) Frequencies of NP-specific ASCs in the spleens from immunized mice with indicated genotypes were analyzed by ELISpot assays. Shown are mean (±SEM) frequencies of high-affinity anti-NP (g) IgM, (h) IgG1, and (i) IgG2c. (j) NP-specific GC B cells were analyzed in the spleen from immunized mice with indicated genotypes. Shown are mean (±SEM) frequencies of NP+ cells in GL7+ CD95+ gated B cells from 3 independent replicate experiments. (k, l) Connecting to Fig 5, mitochondrial (mt)DNA content (k) and MitoTracker Green (MTG) labeling of B lymphoblasts generated as for Fig 5 were measured by qPCR or flow cytometry, respectively.

Impact of CB-839 and UK-5099 on glutaminolysis and mitochondrial metabolism.

(a) A schematic illustration of glutaminolysis, its inhibition by CB-839, and GPT2-(mitochondrial alanine aminotransferase, also termed glutamate pyruvate transaminase) catalyzed conversion of glutamate and pyruvate for generation of alanine. PDH, pyruvate dehydrogenase; MPC, mitochondrial pyruvate channel. (b-j) Selected metabolic perturbations identified by metabolomic analysis of activated B cells. For each of three biologically and temporally independent replicate experiments, B cell pools were purified from several mouse spleens, then activated and cultured 2 d as in Fig 5, or under the indicated conditions [or with Fab2’ anti-IgM (1 µg/mL) added, yielding similar results (not shown)]. Shown are results for (b) glutamine, (c) glutamate, (d) alanine, along with results of calculating inferred glutaminase activities (e). (f) Schematic illustrating mitochondrial import of fatty acids via L-carnitine and acyl-carnitine intermediaries, along with use of the acetyl (Ac)-CoA for either entry into the Krebs (TCA) cycle or generation of acetyl (Ac)-carnitine. LC, long-chain; coA, coenzyme A; CPT, carnitine palmitoyl transferase, FAO, fatty acid oxidation; acetyl, Ac; TCA, tricarboxylic acid (Krebs cycle); CACT, carnitine-acylcarnitine transferase; CrAT, carnitine O-acetyltransferase. (g) Computationally inferred activity of fatty acid oxidation (FAO) derived from the metabolomic data. (h) L-carnitine and (i) acetyl-carnitine concentrations in the activated B cells, and the ratio calculated for each sample (j).

Glutaminolysis enhances rates of Ab secretion by PC in addition to promoting their development.

Using conditions akin to Fig. 4a-e, 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,

Combinatorial reduction in PC differentiation k hydroxychloroquine and glutaminolysis inhibition includes a division-independent mcchanism.

(a) Plasma cell differentiation cultures of purified B cells, labelled with CTV and activated in the presence of the indicated combinations of drugs (or DMSO vehicle), were performed as in Fig. 4 and analyzed by flow cytometry. (a) Mean (±SEM) %CD138+ cells (day 5) at levels of CTV fluorescence representing divisions 3 through 7 are plotted separately for each condition shown in the key. Open symbols, HCQ added; filled symbols - no HCQ. Line colors are coded as in Fig. 4, 5. Mean results derived from three biologically independent experiments. (b) Quantitative data on frequencies of plasma cells after independent cultures of purified B cells were performed and analyzed as in Fig. 4 (no CTV labeling), in the indicated combinations of drugs. Dots denote individual values for four independent B cell pools and cultures, with bars representing the mean % CD138+. (c, d) HCQ effect on PC development contingent on inhibition of glutaminolysis. Using data from (a), the % CD138+ for each indicated condition in each independent experiment was measured in the CTV peaks representing viable cells that divided four (c) and five (d) times. In each case, a ratio of control to HCQ-treated value was calculated for the condition (DMSO or CB-839 present) (left graph) and the actual % CD138+ with and without HCQ (right graph). (e) qPCR measurement of mtDNA after activation and culture (2 d) as in Fig. 5c. (f) Cellular mitochondrial content determined by MitoTracker Green (MTG) labelling quantified by flow cytometry. Shown are MFI values from each independent experiment after activation and culture (2 d) as in Fig. 5c, then normalized to DMSO-treated condition in each experiment.

Altered gene expression of metabolically reprogrammed B cells

Additional data relating to the analyses of RNA-seq results with flow-purified GC B cells (controls versus those with disruption of Gls, Mpc2, or both; Fig. 4j-l; Fig. 6a-c). (a) In the bubble plot summarizing the results of GSEA using the RNA-seq data, the heat-mapped color coding of each circle denoted the normalized enrichment score on the scale to the right, while the size of each circle indicates the adjusted P value. (b-d) Selected GSEA plots illustrative of the changes in transcriptional programs of GC B cells with altered metabolism due to post-maturation disruption of the genes Gls, Mpc2, or both, as summarized in (a). (b) Enrichment of Myc- and E2F-upregulated mRNA in WT samples as compared to Gls Δ/Δ, Mpc2 Δ/Δ. (c) GLS-dependent increases in expression of RNA of the oxidative phosphorylation and E2F pathways, with GSEA comparing Mpc2 Δ/Δ to Gls Δ/Δ, Mpc2 Δ/Δ GC B cells shown. (d) MPC-dependent increases in expression of RNA of the IFN-γ response and apoptosis program gene sets compared for Gls Δ/Δ versus Gls Δ/Δ, Mpc2 Δ/Δ GC B cells shown.

Normal IFN-R expression yet decreased P-STAT1 in metabolically reprogrammed B cells

(a, b) Deletion efficiency of Gls1 and Mpc2 in vivo. CD19+ IgD+ naïve B cells were flow-purified from spleens of tamoxifen-injected and SRBC-immunized hu-CD20-CreERT2 mice (Glsf/f, Mpc2f/f, or wild-type). Shown are the levels of Gls- (a) and Mpc2-encoded RNA (b) in the cells with the indicated genotypes relative to WT control after normalized to β-actin as an internal control. (c) Immunoblot analysis of whole cell extracts of B cells of the indicated genotypes, purified from tamoxifen-treated mice (two of each genotype probed with anti-GLS and anti-cyclophilin B. (e, f) Active metabolism via GLS1 and MPC2 promotes interferon activation of STAT1. WT or Gls1Δ/Δ; Mpc2Δ/Δ B cells were activated with anti-CD40 and BAFF for 2 days followed by IFN-β (c) or IFN-γ (d) treatment for 15 min. Shown are the representative western blot images from more than three independent experiments. (f) Cell surface IFNAR signal on B cells activated and cultured as in Fig 6 was determined by flow cytometry. Shown is a representative result from replicate experiments (n = 3) analyzing IFNAR expression on viable B cells.