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. 2018 Feb 5;215(2):399–413. doi: 10.1084/jem.20170771

Figure 2.

Figure 2.

B1a B cells have active glycolysis and OXPHOS and low metabolic flexibility in vivo. (A) Representative histogram of Glut1.RBD.GFP staining in peritoneal B1a and Fo B2 B cells and quantification of geometric mean fluorescence intensity (gMFI) of Glut1.RBD.GFP staining in B1a and B2 cells from the spleen and peritoneum. n = 5 WT C57BL/6 individual mice per group. One-way ANOVA with Dunnett correction for multiple testing used. FMO, fluorescence minus one. (B) Representative histogram of 2-NBDG uptake in peritoneal B1a and Fo B2 B cells and gMFI of 2-NBDG in splenic Fo B2 cells and peritoneal B1a B cells. n = 5 WT C57BL/6 individual mice per group. Unpaired Student’s t test used. (C) Basal extracellular acidification rate (ECAR) of peritoneal total B1 B cells (B1a and B1b) compared with Fo B2 cells. Data are n = 5 technical replicates of cells sorted from pools of 10 WT C57BL/6 mice. Unpaired Student’s t test used. (D) Basal oxygen consumption rate (OCR) of peritoneal total B1 B cells (B1a and B1b) compared with Fo B2 B cells. Data are n = 5 technical replicates of cells sorted from pools of 10 WT C57BL/6 mice. Unpaired Student’s t test used. (E) Heat map of relative abundance of polar metabolites extracted from peritoneal total B1 (CD19+CD23−) and splenic Fo B2 B cells (CD19+CD23+) sorted by flow cytometry. Each data point represents cells sorted from pools of five to seven 6-wk-old WT C57BL/6 mice. Heat map coloring is based on row Z score. 2-HG, 2-hydroxyglutarate; IMP, inosine monophosphate; S7P, sedoheptulose-7-phosphate; 3-PG, 3-phosphoglycerate; α-KG, α-ketoglutarate; UMP, uracil monophosphate. (F) Heat map of fractional contribution of 13C to metabolites in the PPP and the TCA cycle after incubation of cells described in E with [U-13C]glucose for 1 h. Heat map coloring is based on row Z score. (G) Fractional contribution (FC) of 13C to metabolites in the TCA cycle after incubation of cells described in E with [U-13C-glucose] for 1 h. Unpaired Student’s t test p-value is adjusted for multiple testing using FDR method (q-value; 5% threshold). (A–G) Data representative of two independent experiments. (H) Experimental schematic. WT C57BL/6 mice were injected i.p. with 2-DG or PBS (control) each day for 7 d. The mechanistic target of 2-DG is illustrated in red. (I) Effect of 2-DG or PBS on relative cellularity of indicated B cell subset at specified location. Each data point represents an individual mouse. n = 5 biological replicates. P-values generated by ANOVA are relative to control, with Dunnett correction for multiple comparisons. Representative of three independent experiments. Mean ± SEM is depicted.