Abstract:B-cell development in the bone marrow comprises proliferative and resting phases in different niches. We asked whether B-cell metabolism relates to these changes. Compared to pro B and small pre B cells, large pre B cells revealed the highest glucose uptake and ROS but not mitochondrial mass, whereas small pre B cells exhibited the lowest mitochondrial membrane potential. Small pre B cells from Rag1;33.C9 μ heavy chain knock-in mice revealed decreased glycolysis (ECAR) and mitochondrial spare capacity compared… Show more
“…Interestingly, basal OCR of stimulated B cells was almost identical to the maximal OCR of unstimulated B cells (0.717 and 0.684 fmoles O 2 /min/cell, respectively). Both subsets of B cells had significant spare respiratory capacity (SRC), which sharply contrasts with activated T cells and naive pre- and pro-B cells that exhibit minimal SRC (Stein et al., 2017, van der Windt et al., 2012). Our data suggest distinct mechanisms of metabolic adaptation for T, immature B, and mature activated B cells.Figure 2B Cell Activation Induces Increases in OXPHOS and the TCA Cycle(A) GSEA on RNA-seq transcriptome data from naive and 24 hr stimulated B cells shows enrichment for OXPHOS and TCA cycle metabolic transcripts.(B) Seahorse extracellular flux analysis measurement of oxygen consumption rate (OCR) in naive and stimulated B cells ( n = 4).(C) UHPLC-MS quantification of log 2 converted average levels of TCA cycle metabolites between stimulated and naive B cells ( n = 3).(D) Isotopomer distribution of labeled glucose for TCA cycle metabolites in naive and stimulated B cells ( n = 3).…”
SummaryB lymphocytes provide adaptive immunity by generating antigen-specific antibodies and supporting the activation of T cells. Little is known about how global metabolism supports naive B cell activation to enable an effective immune response. By coupling RNA sequencing (RNA-seq) data with glucose isotopomer tracing, we show that stimulated B cells increase programs for oxidative phosphorylation (OXPHOS), the tricarboxylic acid (TCA) cycle, and nucleotide biosynthesis, but not glycolysis. Isotopomer tracing uncovered increases in TCA cycle intermediates with almost no contribution from glucose. Instead, glucose mainly supported the biosynthesis of ribonucleotides. Glucose restriction did not affect B cell functions, yet the inhibition of OXPHOS or glutamine restriction markedly impaired B cell growth and differentiation. Increased OXPHOS prompted studies of mitochondrial dynamics, which revealed extensive mitochondria remodeling during activation. Our results show how B cell metabolism adapts with stimulation and reveals unexpected details for carbon utilization and mitochondrial dynamics at the start of a humoral immune response.
“…Interestingly, basal OCR of stimulated B cells was almost identical to the maximal OCR of unstimulated B cells (0.717 and 0.684 fmoles O 2 /min/cell, respectively). Both subsets of B cells had significant spare respiratory capacity (SRC), which sharply contrasts with activated T cells and naive pre- and pro-B cells that exhibit minimal SRC (Stein et al., 2017, van der Windt et al., 2012). Our data suggest distinct mechanisms of metabolic adaptation for T, immature B, and mature activated B cells.Figure 2B Cell Activation Induces Increases in OXPHOS and the TCA Cycle(A) GSEA on RNA-seq transcriptome data from naive and 24 hr stimulated B cells shows enrichment for OXPHOS and TCA cycle metabolic transcripts.(B) Seahorse extracellular flux analysis measurement of oxygen consumption rate (OCR) in naive and stimulated B cells ( n = 4).(C) UHPLC-MS quantification of log 2 converted average levels of TCA cycle metabolites between stimulated and naive B cells ( n = 3).(D) Isotopomer distribution of labeled glucose for TCA cycle metabolites in naive and stimulated B cells ( n = 3).…”
SummaryB lymphocytes provide adaptive immunity by generating antigen-specific antibodies and supporting the activation of T cells. Little is known about how global metabolism supports naive B cell activation to enable an effective immune response. By coupling RNA sequencing (RNA-seq) data with glucose isotopomer tracing, we show that stimulated B cells increase programs for oxidative phosphorylation (OXPHOS), the tricarboxylic acid (TCA) cycle, and nucleotide biosynthesis, but not glycolysis. Isotopomer tracing uncovered increases in TCA cycle intermediates with almost no contribution from glucose. Instead, glucose mainly supported the biosynthesis of ribonucleotides. Glucose restriction did not affect B cell functions, yet the inhibition of OXPHOS or glutamine restriction markedly impaired B cell growth and differentiation. Increased OXPHOS prompted studies of mitochondrial dynamics, which revealed extensive mitochondria remodeling during activation. Our results show how B cell metabolism adapts with stimulation and reveals unexpected details for carbon utilization and mitochondrial dynamics at the start of a humoral immune response.
“…Some conditions may require normalization of MdFI to cell volume by adjusting MdFI to FSC pulse width, which is the preferable parameter to evaluate cell size rather than the height or area of FSc or SSc . For instance, large pre B cells that consume more glucose stain ∼40% brighter with 6‐NBDG but are also significantly larger than small pre B cells . Normalization of 6‐NBDG fluorescence to cell size within these very related cell populations ruled out staining artifacts .…”
These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer‐reviewed by leading experts in the field, making this an essential research companion.
“…13 Consistent with their different rates of proliferation, pro-B cells, large and small pre-B cells, and immature B cells differ significantly in their metabolic profile. 14,15 To foster cell growth, IL-7 induces activation of signaling molecules such as mechanistic target of rapamycin complex 1 (mTORC1) and c-Myc, which drive an anabolic metabolic program 15,16 indispensable for B cell development ( Figure 1). Both mTORC1 and c-Myc are known metabolic master regulators, which are activated in response to mitogenic signals.…”
Section: Me Taboli C Bal An Ce In B Cell Precur Sor Smentioning
In response to mitogenic stimulation, B cells activate different pro-anabolic signaling pathways such as c-Myc-and mTORC1-dependent networks to satisfy the energetic demands of biomass synthesis and proliferation. In order to preserve viability and function, cell growth cannot progress unchecked and must be adjusted according to the availability of nutrients. Nutrient-sensing proteins such as AMPK antagonize mTORC1 activity in response to starvation. If pro-anabolic signaling pathways are aberrantly activated, B cells may lack the metabolic capacity to accommodate their energetic needs, which can lead to cell death. On the other hand, metabolic hyperactivation is a salient feature of cancer cells, suggesting that mechanisms exist, which allow B cells to cope with metabolic stress. The aim of this review is to discuss how B cells respond to a mismatch between energy supply and demand and what the consequences are of metabolic dysregulation in normal and malignant B cells.
K E Y W O R D Sanabolism, autophagy, B cells, metabolic stress, mTORC1, senescence
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