Journal: bioRxiv

Article Title: Plasma cell differentiation, antibody quality, and initial germinal center B cell population depend on glucose influx rate

doi: 10.1101/2023.09.13.557599

Figure Lengend Snippet: (A) CD69, MHC-II, CD80, and CD86 surface expression on wild-type B cells after activation by LPS and culture (4, 20, and 40 hr, as indicated) in BAFF, IL-4, and IL-5, in the absence or presence of glucose, as indicated. (B) CD98 surface expression on B cells 4, 20, and 40h after LPS, BAFF, IL-4, and IL-5 stimulation. Shown are the aggregate results of two independent experiments with five total replicates. * p < 0.05, *** p < 0.001 (Multiple comparisons t-tests). (C) Surface GLUT1 expression is diminished on Slc2a1 Δ / Δ (cKO) B cells. Representative flow plot of surface GLUT1 (Alomone Lab) on CD19 + B cells were from tamoxifen-treated WT ( hCD20 -CreER T2 ) and Glut1 cKO B ( Slc2a1 f/f ; hCD20 -CreER T2 ) mice. (D) Frequencies of BrdU + cells in IgD − GL7 + CD38 neg B cells. Tamoxifen-treated WT ( hCD20 -CreER T2 ) and Glut1 cKO B ( Slc2a1 f/f ; hCD20 -CreER T2 ) mice were immunized with SRBC and harvested 7 days later. Mice were injected twice with BrdU (100 mg/kg i.v.), 1 d and 4 h before harvest.

Article Snippet: In brief, 3 x 10 6 splenocytes were stained with anti-B220, -GL7, -Fas, -IgD, -CD38, NP-APC and a dump channel containing anti-CD11b, -CD11c, -F4/80, -Gr-1, and viability marker (7-AAD or Ghost-Brilliant Violet 510), in selected instances with FITC-anti-human GLUT1 (Alomone Labs, Jerusalem, Israel) in 1% BSA and 0.05% sodium azide in PBS.

Techniques: Expressing, Activation Assay, Injection

Journal: bioRxiv

Article Title: Plasma cell differentiation, antibody quality, and initial germinal center B cell population depend on glucose influx rate

doi: 10.1101/2023.09.13.557599

Figure Lengend Snippet: (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 CreER T2 mice ( Slc2a1 f/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 CreER T2 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 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 Slc2a1 Δ/Δ , designated Glut1 Δ/Δ in earlier work ] after culture (2 d) with LPS, BAFF, and 4-hydroxytamoxifen. Data represent two independent experiments with wild-type and Slc2a1 Δ/Δ 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 GLUT1 lo versus GLUT1 hi 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 Slc2a1 Δ/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 Slc2a11 Δ/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 + B220 lo cells after activation and culture as in (D). (H) Shown are the frequencies of CD138 + B220 lo cells at each cellular division 4 days after activation. Data are aggregated from three independent replicate experiments using wild-type (n = 7) and Slc2a1 Δ/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.

Article Snippet: In brief, 3 x 10 6 splenocytes were stained with anti-B220, -GL7, -Fas, -IgD, -CD38, NP-APC and a dump channel containing anti-CD11b, -CD11c, -F4/80, -Gr-1, and viability marker (7-AAD or Ghost-Brilliant Violet 510), in selected instances with FITC-anti-human GLUT1 (Alomone Labs, Jerusalem, Israel) in 1% BSA and 0.05% sodium azide in PBS.

Techniques: In Vivo, Activation Assay, In Vitro, Purification, Cell Culture, Extraction, Expressing, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: Plasma cell differentiation, antibody quality, and initial germinal center B cell population depend on glucose influx rate

doi: 10.1101/2023.09.13.557599

Figure Lengend Snippet: (A) Schematic depicting immunization with SRBC after inactivation of Slc2a1 in mature B cells. Mice (huCD20-CreER T2 , Slc2a1 +/+ or CreER T2 , Slc2a1 f/f ) were harvested 1 wk after immunization. Representative flow plots of viable splenic IgD neg B cells (B) and aggregate data for two replicate experiments (C) (n=2 for each genotype in each). (D) In vivo BrdU incorporation into IgD neg B cells of the mice in (A-C). Shown are aggregate data, with representative flow plots in . (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 (CreER T2 , Slc2a1 +/+ or CreER T2 , Slc2a1 f/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 (G). (I) Relative concentrations of circulating NP-specific IgM and IgG1 in the sera of wild-type and Slc2a1 Δ/Δ mice one week after NP-ova immunization determined by ELISA. Data represent 4 independent experiments with wild-type (n = 13) and Slc2a1 Δ/Δ (n=14) mice. Probabilities of the null hypothesis being correct were * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: In brief, 3 x 10 6 splenocytes were stained with anti-B220, -GL7, -Fas, -IgD, -CD38, NP-APC and a dump channel containing anti-CD11b, -CD11c, -F4/80, -Gr-1, and viability marker (7-AAD or Ghost-Brilliant Violet 510), in selected instances with FITC-anti-human GLUT1 (Alomone Labs, Jerusalem, Israel) in 1% BSA and 0.05% sodium azide in PBS.

Techniques: In Vivo, BrdU Incorporation Assay, Purification, Binding Assay, Produced, Enzyme-linked Immunospot, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Plasma cell differentiation, antibody quality, and initial germinal center B cell population depend on glucose influx rate

doi: 10.1101/2023.09.13.557599

Figure Lengend Snippet: (A) Schematic model of adoptive transfer and immunization to test the effect of GC B cell-specific role of GLUT1. B cells from S1pr2 -CreER T2 mice (wild-type or Slc2a1 f/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) Representative flow plots (B), quantified frequencies of Fas + GL7 + cells in the B220 + IgD neg Dump neg 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. (E) qPCR measurement of Slc2a1 floxed allele in freshly sorted CD19 + IgD − GL7 + CD38 − GC B cells from tamoxifen-treated WT ( S1pr2 -CreER T2 ) and Glut1 cKO GCB ( Slc2a1 f/f ; S1pr2 -CreER T2 ) mice. (F) Numbers of all-affinity NP-specific IgM [a] and IgM [b] ASCs that derived from wild-type (•) and GCB-specific Slc2a1 Δ/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 -CreER T2 B cell recipients and n = 8 mice that received GC-specific Slc2a1 Δ/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.

Article Snippet: In brief, 3 x 10 6 splenocytes were stained with anti-B220, -GL7, -Fas, -IgD, -CD38, NP-APC and a dump channel containing anti-CD11b, -CD11c, -F4/80, -Gr-1, and viability marker (7-AAD or Ghost-Brilliant Violet 510), in selected instances with FITC-anti-human GLUT1 (Alomone Labs, Jerusalem, Israel) in 1% BSA and 0.05% sodium azide in PBS.

Techniques: Adoptive Transfer Assay, Irradiation, Derivative Assay

Journal: bioRxiv

Article Title: Plasma cell differentiation, antibody quality, and initial germinal center B cell population depend on glucose influx rate

doi: 10.1101/2023.09.13.557599

Figure Lengend Snippet: (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 Slc2a1 from 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 + IgD neg GL7 neg CD38 ++ ) B cells at harvest. (C) Left panel, representative ELISpot wells (5 x 10 5 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 Slc2a1 Δ/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 (NP 2 –PSA). (F) Affinity maturation of IgG1 and IgG2c Ab, calculated as ratios of high-affinity (captured on NP 2 –PSA) to all-affinity (captured on NP 27 –BSA) OD 450 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 Slc2a1 Δ/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).

Article Snippet: In brief, 3 x 10 6 splenocytes were stained with anti-B220, -GL7, -Fas, -IgD, -CD38, NP-APC and a dump channel containing anti-CD11b, -CD11c, -F4/80, -Gr-1, and viability marker (7-AAD or Ghost-Brilliant Violet 510), in selected instances with FITC-anti-human GLUT1 (Alomone Labs, Jerusalem, Israel) in 1% BSA and 0.05% sodium azide in PBS.

Techniques: Enzyme-linked Immunospot, Quantitation Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: Plasma cell differentiation, antibody quality, and initial germinal center B cell population depend on glucose influx rate

doi: 10.1101/2023.09.13.557599

Figure Lengend Snippet: (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 Slc2a1 Δ/Δ ) were assayed after activation and culture (2 d) with LPS, BAFF, IL-4, and IL-5. (C) Glycolytic reserve of wild-type and Slc2a1 Δ/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-[ 14 C]-glucose and 6-[ 14 C]-glucose conversion to 14 CO 2 in the indicated flow-purified splenocyte populations after SRBC immunization. (E) PPP activity determined by 1-[ 14 C]-glucose conversion to 14 CO 2 after short-term cultures of flow purified wild-type and Slc2a1 Δ/Δ B cells (B220 + CD138 neg ) and ASCs (B220 lo CD138 + ). Purified B cells were activated and cultured (4 d) with LPS, BAFF, IL-4, and IL-5. 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.

Article Snippet: In brief, 3 x 10 6 splenocytes were stained with anti-B220, -GL7, -Fas, -IgD, -CD38, NP-APC and a dump channel containing anti-CD11b, -CD11c, -F4/80, -Gr-1, and viability marker (7-AAD or Ghost-Brilliant Violet 510), in selected instances with FITC-anti-human GLUT1 (Alomone Labs, Jerusalem, Israel) in 1% BSA and 0.05% sodium azide in PBS.

Techniques: Generated, Activation Assay, Activity Assay, Purification, Cell Culture

Journal: bioRxiv

Article Title: Plasma cell differentiation, antibody quality, and initial germinal center B cell population depend on glucose influx rate

doi: 10.1101/2023.09.13.557599

Figure Lengend Snippet: (A) Mitochondrial reactive oxygen species in different B cell subsets measured by ex vivo MitoSOX staining and flow cytometry using mice (wildtype and Slc2a1 Δ/Δ ) harvested one week after NP-ova immunization. (B, C) Total cellular ROS (B) and (C) mitochondrial ROS in B lymphoblasts determined by H 2 DCFDA 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.

Article Snippet: In brief, 3 x 10 6 splenocytes were stained with anti-B220, -GL7, -Fas, -IgD, -CD38, NP-APC and a dump channel containing anti-CD11b, -CD11c, -F4/80, -Gr-1, and viability marker (7-AAD or Ghost-Brilliant Violet 510), in selected instances with FITC-anti-human GLUT1 (Alomone Labs, Jerusalem, Israel) in 1% BSA and 0.05% sodium azide in PBS.

Techniques: Ex Vivo, Staining, Flow Cytometry, Fluorescence, Activation Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Plasma cell differentiation, antibody quality, and initial germinal center B cell population depend on glucose influx rate

doi: 10.1101/2023.09.13.557599

Figure Lengend Snippet: (A) Schematic of immunization strategy to test B cell loss of Slc2a1 in a lung inflammation model. Mice were primed with two I.P. injections of NP-ova, followed by sequential ovalbumin inhalations received daily for seven days starting 3 wk after initial immunization and continuing until harvest. (B) Frequency of GCB in mediastinal lymph nodes of wildtype and Slc2a1 Δ/Δ mice at the time of harvest, as determined by flow cytometry. (C) Left panel , representative ELISpot wells of ovalbumin-specific IgG1-secreting cells in the lung (among 10 6 cells in lung dispersion) and bone marrow (2 x 10 6 bone marrow cells plated/well) of mice with wild-type or Slc2a1 Δ/Δ B cells. Right panel , numbers of ovalbumin-specific IgG1 antibody-secreting cells in the lung or bone marrow per 10 5 total cells. (D) As in (C) except ELISpot captured NP-specific ASC. Left panel , representative ELISpot wells visualizing IgG1-secreting bone marrow cells from immunized mice with wild-type or Slc2a1 Δ/Δ B cells. Right panel , numbers of NP-specific IgG1 ASC in the bone marrow per 10 6 cells. (E) Ovalbumin-specific IgE (left panel) and IgG1 (right panel) in unimmunized, wild-type, and Slc2a1 Δ/Δ mice at the terminal time point depicted as OD 450nm values after three or two-fold serial dilutions of the sera for ELISA, respectively. (F) NP-specific IgM in the sera of mice at week 3, prior to inhaled challenges with ovalbumin. As for (E), except serial dilutions were 4-fold. (G) All-affinity (left panel) and high affinity (right panel) NP-specific IgG1 in the sera of mice at week 3, prior to inhaled challenges with ovalbumin. (H) Shown are the ratios of OD values of NP 2 -bound IgG1 antibodies (high affinity) measured in ELISA to those of NP 27 -bound (all affinity) IgG1; ratios were calculated at a dilution (1:12,800) with values for both WT and GLUT1-deficient B cells in the linear range. Data are representative of two independent experiments with n = 7 wild-type and n = 4 Slc2a1 Δ/Δ mice. * p < 0.05, ** p < 0.01. Mann Whitney U (B-D, H), Two-way ANOVA (E, G).

Article Snippet: In brief, 3 x 10 6 splenocytes were stained with anti-B220, -GL7, -Fas, -IgD, -CD38, NP-APC and a dump channel containing anti-CD11b, -CD11c, -F4/80, -Gr-1, and viability marker (7-AAD or Ghost-Brilliant Violet 510), in selected instances with FITC-anti-human GLUT1 (Alomone Labs, Jerusalem, Israel) in 1% BSA and 0.05% sodium azide in PBS.

Techniques: Flow Cytometry, Enzyme-linked Immunospot, Dispersion, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY