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. 2024 Jan 1;212(1):43-56.
doi: 10.4049/jimmunol.2200756.

Plasma Cell Differentiation, Antibody Quality, and Initial Germinal Center B Cell Population Depend on Glucose Influx Rate

Shawna K Brookens  1   2   3 , Sung Hoon Cho  1   4   5 , Yeeun Paik  1 , Kaylor Meyer  1 , Ariel L Raybuck  1 , Chloe Park  1 , Dalton L Greenwood  1 , Jeffrey C Rathmell  1   2   4   5 , Mark R Boothby  1   2   4   5
Affiliations

Plasma Cell Differentiation, Antibody Quality, and Initial Germinal Center B Cell Population Depend on Glucose Influx Rate

Shawna K Brookens et al. J Immunol. .

Abstract

Serum Ab concentrations, selection for higher affinity BCRs, and generation of higher Ab affinities are important elements of immune response optimization and functions of germinal center (GC) reactions. B cell proliferation requires nutrients to support the anabolism inherent in clonal expansion. Glucose usage by mouse GC B cells has been reported to contribute little to their energy needs, with questions raised as to whether glucose uptake or glycolysis increases in GC B cells compared with their naive precursors. Indeed, metabolism can be highly flexible, such that supply shortage along one pathway may be compensated by increased flux on others. We now show that reduction of the glucose transporter GLUT1 in mice after establishment of a preimmune B cell repertoire, even after initiation of the GC B cell gene expression program, decreased initial GC B cell population numbers, affinity maturation, and plasma cell outputs. Glucose oxidation was heightened in GC B cells, but this hexose flowed more into the pentose phosphate pathway, whose activity was important in controlling reactive oxygen species (ROS) and Ab-secreting cell production. In modeling how glucose usage by B cells promotes the Ab response, the control of ROS appeared insufficient. Surprisingly, the combination of galactose, which mitigated ROS, with provision of mannose, an efficient precursor to glycosylation, supported robust production of and normal Ab secretion by Ab-secreting cells under glucose-free conditions. Collectively, the findings indicate that GCs depend on normal glucose influx, especially in plasma cell production, but reveal an unexpected metabolic flexibility in hexose requirements.

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Figures

Figure 1.
Figure 1.. Activation and developmental stage-dependent differences in requisite glucose uptake.
(A) Left, schematic depicting immunization with NP-ova in alum to allow measurements of multiple B lineage cell types within the same mouse. Right panel, ex vivo 2-NBDG signal in splenic populations: naïve B cells (B220+ IgD+ GL7neg), antigen-specific germinal center B cells (GCB; B220+ GL7+ IgDneg NP+), antigen-specific memory B cells (MBC; B220+ CD38+ IgD− NP+) and antibody secreting cells (ASC; CD138+). Each dot shows the mean fluorescence intensity in the indicated cell-type gate, with mean (±SEM) values among the samples (n=8) presented as bar graph. (B) Glucose uptake into naïve (IgD+, GL7neg, CD138neg) and germinal center B cells (IgDneg, GL7+, CD138neg) purified by flow cytometry one week after SRBC immunization, measured using 2-[1,2-3H]-deoxyglucose. Each dot shows the measured counts, with mean (±SEM) values among the samples (n=6) as in (A). (C) Glucose uptake into in vitro-generated B lymphoblasts, measured using 2-[1,2-3H]-deoxyglucose 2 days after activation with LPS and BAFF. Data are shown as in (B). (D) Left panel, BrdU incorporation into DNA of viable cells after culture (2 d) of B lymphoblasts activated with LPS and grown in the presence or absence of glucose (10 mM) in a glucose-free medium supplemented with dialyzed FBS (10%), BAFF, IL-4, and IL-5. Right panel, representative flow cytometric analysis of BrdU incorporation. Data represent four independent experiments totaling 15 B cell preparations from separate mice. (E) Cell Trace Violet (CTV) partitioning four days after activation with LPS and culture in BAFF, IL-4 and IL-5 in the presence or absence of glucose as indicated. (F) ASC differentiation (CD138 expression) as a function of CTV partitioning four days after activation and culture as in (E). (G) IgG1 concentration in the supernatants of these 4 day cultures. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Mann-Whitney U test.
Figure 2.
Figure 2.. Sufficiency of glucose influx is critical for plasmablast differentiation in vitro.
(A, B) Deletion efficiency of Slc2a1 fl alleles in vivo (A) and after activation and culture (2 d) in vitro (B). (A) Naïve-phenotype B cells were flow-purified from spleens of CreERT2 mice (Slc2a1f/f and wild-type) after in vivo tamoxifen injections. Shown are both the extent of deletion (bar graph to left, i.e., reduction of the signal for the fl allele in qPCR measurements of DNA from Slc2a1 f/f B cells after tamoxifen injections in CreERT2 mice), and the level of Slc2a1-encoded RNA (right panel). and culture (2 d) with LPS, BAFF, and 4-hydroxytamoxifen. (B) Flow-purified naïve B cells from experiments in (A) were activated cultured 2 d in BAFF and LPS, followed by DNA and RNA purification and q(RT2)PCR to quantitate Slc2a1 sequences as in (A). Left and right bar graphs are as in (A). Each shows data from four (4) mice of each genotype (non-recombined / deleted vs deleted) in two biologically independent replicate experiments of 2 vs 2 mice each, with p values calculated by unpaired Student’s t-test. (C) Glucose consumption (extraction from culture media) determined by H+ NMR measurements using the supernatants of B lymphoblasts [wild-type vs Slc2a1Δ/Δ, designated Glut1Δ/Δ in earlier work (12)] after culture (2 d) with LPS, BAFF, and 4-hydroxytamoxifen. Data represent two independent experiments with wild-type (5) and Slc2a1Δ/Δ (7) mice. (D) Flow cytometric measurement of CTV partitioning of wild-type and GLUT1-deficient B220+ cells 4 d after activation and culture as in (A). (E, F) GLUT1 expression level influences CD138+ generation in vitro. Naïve B cells, separated as GLUT1lo versus GLUT1hi using an ectodomain-directed anti-GLUT1, were flow-purified, followed by activation and culture (5 d) in LPS, BAFF, IL-4, and IL-5. (E) Shown are representative flow plots of CD138 staining at the end of cultures, with inset numbers providing the percentages of CD138+ events from the WT and Slc2a1Δ/Δ B cells. (F) Quantified frequencies of CD138+ cells after the cultures as in (F), starting from flow-purified B cells of the wild-type (n=4) and B cell-specific Slc2a11Δ/Δ gene inactivated (n = 4) mice in two independent experiments. p values were calculated by unpaired Student’s t-test. (G) Representative plot (left) and quantification (right) of the frequencies of CD138+ B220lo cells after activation and culture as in (D). (H) Shown are the frequencies of CD138+ B220lo cells at each cellular division 4 days after activation. Data are aggregated from three independent replicate experiments using wild-type (n = 7) and Slc2a1Δ/Δ (n = 8) mice. The probabilities that the null hypothesis would be correct were * p < 0.05, ** p < 0.01, *** p < 0.001 by Mann-Whitney U testing.
Figure 3.
Figure 3.. Early immunization-induced germinal center formation, ASC development, and Ab production in vivo require normal glucose transport.
(A) Schematic depicting immunization with SRBC after inactivation of Slc2a1 in mature B cells. Mice (huCD20-CreERT2, Slc2a1+/+ or CreER T2, Slc2a1f/f) were harvested 1 wk after immunization. Representative flow plots of viable splenic IgDneg B cells (B) and aggregate data for two replicate experiments (C) (n=2 for each genotype in each). (D) In vivo BrdU incorporation into IgDneg B cells of the mice in (A-C). Shown are aggregate data, with representative flow plots in Supplemental Fig. 1D. (E) Shown are PCR results for the Slc2a1 fl allele in flow-purified GC B cells of the mice in (A-C). (F) Schematic depicting immunization with NP-ova after inactivation of Slc2a1 in mature B cells. Mice (CreERT2, Slc2a1+/+ or CreER T2, Slc2a1f/f) were harvested 1 wk after immunization. (G) Representative plots (left panel) and total numbers of splenic Ag-binding GCB (right panel) after a single immunization as in (F). Each dot represents an independent mouse of the indicated genotype, with the mean (±SEM) values depicted by bar graph. (H) Ab-secreting cells (ASC) that produced NP-binding IgM and IgG1 one week after immunization were enumerated by ELISpot assays with spleen cell suspensions of the NP-ova-immunized mice as in (F, G). (I) Relative concentrations of circulating NP-specific IgM and IgG1 in the sera of wild-type and Slc2a1Δ/Δ mice one week after NP-ova immunization determined by ELISA. Data represent 4 independent experiments with wild-type (n = 13) and Slc2a1Δ/Δ (n=14) mice. Probabilities of the null hypothesis being correct were * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Figure 4.
Figure 4.. GLUT1 expression within GC B cells regulates their prevalence and PC generation.
(A) Schematic model of adoptive transfer and immunization to test the effect of GC B cell-specific role of GLUT1. B cells from S1pr2-CreERT2 mice (wild-type - Slc2a1+/+ - or Slc2a1f/f) were mixed with CD45.1; IgH[a] B cells and CD4 T cells, then transferred into irradiated CD45.1; IgH[a] recipient mice followed by immunization with NP-ova. Starting 5 d thereafter, mice were treated with tamoxifen and harvested at day 14. (B-D) Flow plots (B), quantified frequencies of Fas+ GL7+ cells in the B220+ IgDneg Dumpneg gate (Dump: CD11b, CD11c, F4/80, Gr1, 7-AAD) after gating to distinguish CD45.1 versus CD45.2 (C), and (D) the ratios of CD45.2 GCB / CD45.1 GC B cells are shown. (E) Numbers of all-affinity NP-specific IgM[a] and IgM[b] ASCs that derived from wild-type () and GCB-specific Slc2a1Δ/Δ (°) B cells (left), and the ratios of NP-specific IgM[b] ASC/ NP-specific IgM[a] ASC harvested from each recipient (right) (n = 8 wild-type S1pr2-CreERT2 B cell recipients and n = 8 mice that received GC-specific Slc2a1Δ/Δ B cells in three independent experiments). P values for the likelihood the null hypothesis is correct were calculated by unpaired Student’s t-test. (F) qPCR measurement of Slc2a1 floxed allele in freshly sorted CD19+ IgD− GL7+ CD38− GC B cells from tamoxifen-treated WT (S1pr2-CreERT2) and Glut1 cKOGCB (Slc2a1f/f; S1pr2-CreERT2) mice.
Figure 5.
Figure 5.. Glucose flux into B lineage cells ASC generation and the production of higher affinity antibodies and restrains early MBC formation.
(A) Schematic depicting prime-boost hapten-carrier immunizations after depletion of Slc2a1 from mature B cells. Mice immunized with NP-ova after tamoxifen-induced conversion of Slc2a1from f/f to Δ/Δ in mature B cells were harvested at week 4, one week after a booster immunization. (B) Total numbers of splenic NP+ memory-phenotype (B220+ IgDneg GL7neg CD38++) B cells at harvest. (C) Left panel, representative ELISpot wells (5 × 105 splenocytes seeded per well) for quantitation of splenic ASCs secreting NP-specific IgM and IgG1 (all- and high-affinity), as indicated. Right panel, splenic ASC prevalence in individual mice, with each dot representing an individual subject. (D) Relative concentrations of all-affinity NP-specific IgM, IgG1, and IgG2c of unimmunized (), wildtype (), and Slc2a1Δ/Δ (°) mice at the time of harvest (1 wk after boost), measured by ELISA across serial four-fold dilutions of the indicated sera. Shown are mean (±SEM) absorbances at each dilution for mice whose B cells were of the indicated genotype. (E) High-affinity NP-specific IgG1 and IgG2c in the sera at the time of harvest, as in (D) but using low-valency NP (NP2–PSA). (F) Affinity maturation of IgG1 and IgG2c Ab, calculated as ratios of high-affinity (captured on NP2–PSA) to all-affinity (captured on NP27–BSA) OD450 ELISA values in the linear range (1:16,000 dilution for IgG1 or 1:200 for IgG2c). Data are representative of wild-type (n = 7) and Slc2a1Δ/Δ (n = 5) mice in two independent experiments. * p < 0.05, ** p < 0.01. Mann-Whitney U test (C), two-way ANOVA (E), Student’s t-test (B, E, F).
Figure 6.
Figure 6.. GLUT1 supports metabolic flux in B lymphoblasts and plasma cells.
(A) Schematic showing two major fates of glucose-6-phosphate (G6P) generated after glucose entry through the GLUT1 transporter, i.e., glycolysis and shunting into the Pentose Phosphate Pathway. (B) Extracellular acidification rates (ECAR) during metabolic flux analyses conducted as Glycolytic Stress Tests of lymphoblasts. B cells (wildtype or Slc2a1Δ/Δ) were assayed after activation and culture (2 d) with LPS, BAFF, IL-4, and IL-5. (C) Glycolytic reserve of wild-type and Slc2a1Δ/Δ B cells calculated from assays in (B). Dots and bar graphing are as in previous figure panels. (D) PPP activity and glucose oxidation determined by 1-[14C]-glucose and 6-[14C]-glucose conversion to 14CO2 in the indicated flow-purified splenocyte populations after SRBC immunization. (E) PPP activity determined by 1-[14C]-glucose conversion to 14CO2 after short-term cultures of flow-purified wild-type and Slc2a1Δ/Δ B cells (B220+ CD138neg) and ASCs (B220lo CD138+). Purified B cells were activated and cultured (4 d) with LPS, BAFF, IL-4, and IL-5, then flow sorted and cultured with 1-[14C]-glucose. Data represent two (B, C) or three (D, E) independent experiments, each with multiple B cell preparations from separate mice. * p < 0.05, ** p < 0.01.
Figure 7.
Figure 7.. Sufficient activity of pentose phosphate pathway is critical for ROS regulation and the generation of antibody secreting cells.
(A) Mitochondrial reactive oxygen species in different B cell subsets measured by ex vivo MitoSOX staining and flow cytometry using mice (wildtype and Slc2a1Δ/Δ) harvested one week after NP-ova immunization. (B, C) Total cellular ROS (B) and (C) mitochondrial ROS in B lymphoblasts determined by H2DCFDA and MitoSOX staining, respectively, followed by flow cytometry. Shown are mean fluorescence intensity (MFI) values for each independent experiment after B cell activation with LPS and culture (2 d) in BAFF, IL-4, and IL-5 in the presence of PPP inhibitors DHEA and 6-AN as indicated. (D) Frequency of CD138+ cells after culture ± PPP inhibitor treatment. Cells were activated and cultured as in (B), followed by flow cytometry. (E) Relative concentrations of IgM and (F) IgG1 in supernatants 5 days after B cell activation and culture (5 d) as in (A-D), in the presence of PPP inhibitors as indicated. Shown are mean (±SEM) ELISA results from three independent experiments and samples, using supernatants at dilutions established as being in the linear range (1:4,000 and 1:1,000 for detection of IgM and IgG1, respectively). * p < 0.05, *** p < 0.001, **** p < 0.0001.
Figure 8.
Figure 8.. Collaborative hexose usage supports plasma cell and efficient antibody production.
(A) Simplified schematic of hexose utilization and monosaccharide metabolism. (B) ECAR and (C) OCR measurements of LPS-activated wild-type B cells cultured (2 d) in BAFF, IL-4, IL-5. Metabolic fluxes of equal numbers of B lymphoblasts were analyzed using either glucose or galactose, as indicated, in ‘glycolytic stress testing’ programmed into the Seahorse XFe. Shown are the hexose-stimulated proton secretion (B), net of basal ECAR in hexose-free base medium, and mitochondrial respiration (C). Data represent four independent experiments with paired t-test comparisons. Representative raw data from a single experiment are shown in Supplemental Fig 3, panels C, D. (D) MFI of total cellular ROS (H2DCFDA) and (E) mitochondrial ROS (MitoSOX) of wild-type B lymphoblasts after activation with LPS and culture (2 d) with BAFF, IL-4, and IL-5 in glucose-free medium supplemented with glucose (Glc), galactose (Gal), Mannose (Man), or both Gal and Man. No hexose, (−). (F) Frequency of CD138+ cells in the live gate determined by flow cytometry after 5 days of culture as above. Results of statistical analyses in (D-F) represent comparison of each condition to the unsupplemented group by the Student’s t-test, with * - **** defined as in Fig 7. (G) Total number of CD138+ cells after provision of hexoses, calculated from the viable cell numbers and frequencies of CD138+ cells measured in (F). (H) Representative ELISpot wells of IgM and IgG1-secreting cells 5 days after cells were cultured in different hexose supplemented media as above and seeding equal numbers of viable cells (100 cells per well). (I) Quantitation of (H), depicting percentage of IgM and IgG1 secreting cells detected among total cells seeded in 5-day hexose cultures. (J) IgM and IgG1 antibody production per cell was analyzed by measurement of average spot sizes in each well. Data aggregate two independent replicate experiments, each with three independent B cell preparations cultured under the indicated conditions and analyzed by ELISpot assays. (G-J) Mann-Whitney U test determined statistical significance and indicated by * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
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