Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2022 Nov 1.
Published in final edited form as: J Immunol. 2022 Apr 6;208(9):2098–2108. doi: 10.4049/jimmunol.2100356

Pharmacologically inferred glycolysis and glutaminolysis requirement of B cells in lupus-prone mice

Seung-Chul Choi 1, Wei Li 1, Xiaojuan Zhang 1, Nathalie Kanda 1, Leilani Zeumer-Spataro 1, Xiangyu Teng 1, Laurence Morel 1,*
PMCID: PMC9050845  NIHMSID: NIHMS1782669  PMID: 35387839

Abstract

Several studies have shown an enhanced metabolism in the CD4+ T cells of lupus patients and lupus-prone mice. Little is known on the metabolism of B cells in lupus. Here we compared the metabolism of B cells between lupus-prone B6.Sle1.Sle2.Sle3 (TC) mice and B6 controls at steady state relative to autoantibody production, as well as during T cell-dependent (TD) and T cell-independent (TI) immunizations. Starting before the onset of autoimmunity, B cells from TC mice showed an elevated glycolysis and mitochondrial respiration, which were normalized in vivo by inhibiting glycolysis with a 2-deoxy-D-glucose (2DG) treatment. 2DG greatly reduced the production of TI-antigen-specific antibodies, but showed minimal effect with TD-antigens. In contrast, the inhibition of glutaminolysis with 6-Diazo-5-oxo-L-norleucine (DON) had a greater effect on TD than TI Ag-specific antibodies in both strains. Analysis of the TI and TD responses in purified B cells in vitro suggests, however, that the glutaminolysis requirement is not B cell-intrinsic. Thus, B cells have a greater requirement for glycolysis in TI than TD-responses, as inferred from pharmacological interventions. B cells from lupus-prone and control mice have different intrinsic metabolic requirement, or different responses towards 2DG and DON. which mirrors our previous results obtained with follicular helper T cells. Overall, these results predict that targeting glucose metabolism may provide an effective therapeutic approach for systemic autoimmunity by eliminating both autoreactive TFH and B cells, although it may also impair TI-responses.

Keywords: B cell, SLE, glycolysis, glutaminolysis, germinal centers

Introduction

B lymphocytes residing in secondary lymphoid tissues are mainly quiescent with low metabolic demands before encountering their cognate antigens. Upon antigen recognition, they differentiate into plasmablasts and plasma cells (here globally referred to as antibody-forming cells, AFCs) through germinal center (GC)-dependent or independent pathways to produce antigen-specific antibodies (1). B cells present different metabolic demands during this process depending on their developmental stage and their location (2). Activated B cells show an increased glycolysis and oxidative phosphorylation (OXPHOS) (3–5), and GC B cells developing from activated B cells retain an increased glucose consumption and mitochondrial mass (6–8). A recent study has however shown that GC B cells induced by protein immunization were solely dependent on fatty acid (FA) oxidation (9), suggesting complex metabolic requirements that may vary with the type of stimuli. Similarly, the final stages of B cell differentiation into AFCs, which correspond to a heterogeneous population in terms of lifespan, ontogeny and anatomical location, also present different metabolic requirements; however, mitochondrial glucose utilization promotes plasma cell survival (10).

Systemic lupus erythematosus (SLE) is a prototypical systemic autoimmune disease in which pathogenic autoantibodies are generated both through GCs (11) and extrafollicular B cell maturation (12). We have shown that CD4+ T cells from SLE patients and mouse models of the disease have an increased glycolysis and mitochondrial metabolism, and that a dual treatment with 2DG, which inhibits glycolysis, and metformin, which inhibits complex 1 of the mitochondrial electron transport chain, reversed disease in mice (13; 14). Glycolysis and mitochondrial metabolism have not been evaluated in the B cells of either patients or mouse models, although it has been shown that an increased AKT/mTOR signaling was a common feature of B cells across several mouse models of lupus (15). Follicular helper T (TFH) cells from lupus-prone mice are highly sensitive to glycolysis inhibition, which also greatly reduced the expansion of GC B cells as well as the production of autoantibodies (16). The induction of TFH and GC B cells by protein or viral immunization in either lupus-prone or control mice was however not dependent on glycolysis. Because of the interdependence of TFH and GC B cell development and function, it is not clear whether the differences between lupus-prone and control strains as well as between auto vs. foreign antigen triggers correspond to an intrinsic glycolysis requirement of autoreactive GC B cells, TFH cells, or both. In addition, the inhibition of glutaminolysis with DON greatly reduced the induction of GC B cells, and to a lesser extent TFH cells, as well as the resulting antibody responses induced by both autoantigen and immunization (16). This indicated that glutaminolysis is required for GC development, but again, the respective intrinsic requirements of GC B and TFH cells for glutaminolysis are unknown. Therefore, we compared the metabolism of B cells between the B6.NZM.Sle1.Sle2.Sle3 triple congenic (TC) model of lupus (17) and its congenic non-autoimmune control C57BL/6 (B6), as well as their response to 2DG and DON at steady state as well as during primary T cell-dependent (TD) and T cell-independent (TI) humoral immune responses. We also conducted these comparisons on purified B cells stimulated in vitro in TI and TD conditions.

Materials and Methods

Mice and in vivo treatment with metabolic inhibitors

C57Bl/6J (B6) were originally obtained from the Jackson Laboratory. B6.NZM-Sle1NZM2410/AegSle2NZM2410/AegSle3NZM2410/Aeg/LmoJ (TC) congenic mice have been previously described (17). All mice were bred and maintained at the University of Florida in specific pathogen-free conditions. For in vivo treatment, 2DG (Sigma, 6 mg/ml) was dispensed in drinking water for the entire duration of each experiment. Age-matched control mice received plain drinking water. DON (Sigma, 1.6 mg/kg) was injected intra-peritoneally every other day for 2 weeks. Rapamycin (RAPA, Sigma 8 ug/kg) was injected daily intra-peritoneally. Control mice received PBS injections. Only female mice were used at the age indicated for each experiment, in which pre-autoimmune and autoimmune stages were defined relative to the production of anti-dsDNA IgG, which starts between 4 and 5 months of age in that strain. All protocols approved by the Institutional Animal Care and Use Committees of the University of Florida.

Flow cytometry

Single-cell suspensions were prepared from spleens using standard procedures. After red blood cell lysis, cells were blocked with anti-CD16/32 Ab (2.4G2), and stained in FACS staining buffer (2.5% FBS, 0.05% sodium azide in PBS). Fluorochrome-conjugated Abs used were to B220 (RA3–6B2), BCL6 (K112–91), CD1d (1B1), CD4 (RAM4–5), CD21/35 (4E3), CD23 (B3B4), CD25 (PC61.5), CD93 (AA4.1), CD95 (Jo2), CD138 (281-2), CD279 (RMP1–30), CXCR5 (2G8), FOXP3 (FJK-16s), GL-7 (GL7), IgDb (217-170), IgM (II/41), and phospho-S6 (D57.2.2E), anti-phospho 4EBP1 (236B4) and anti-phospho Ser 473 AKT1 (SDRNTR) purchased from BD Biosciences, Thermo Fisher, BioLegend, or Cell Signaling Technology. For intracellular staining, cells were fixed and permeabilized with FOXP3 stating buffer (Thermo Fisher). 4-hydroxy-3-nitrophenylacetyl (NP)-phycoerythrin was purchased from Biosearch Technology. In vitro TD and TI cultured B cells (1–2 × 106) for 4 d were incubated with 20 μM 2-N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino-2-Deoxyglucose (2-NBDG) or 2 μM Bodipy (both from Sigma) for 20 min at 37°C to measure glucose or lipid uptake, respectively. Dead cells were excluded with fixable viability dye (eFluor780; Thermo Fisher). Data were collected on LSRFortessa (BD Biosciences) and analyzed with FlowJo software (Tree Star).

TD and TI Immunization and ELISA

8–10 week-old mice received an intra-peritoneal injection of 4-hydroxy-3-nitrophenylacetyl conjugated to keyhole limpet hemocyanin (NP(31)-KLH, 100 μg) or NP-conjugated lipolysaccharide (NP(0.4)-LPS, 50 μg), both purchased from Biosearch Technology, in alum for TD or TI immunization, respectively, and were analyzed 10 d later for TD and 7 d later for TI. Mice were pretreated with 2DG for 2 weeks before immunization, or administered with DON or RAPA starting on the day of immunization. Serum NP-specific antibodies were measured by ELISA using plates coated with NP(4)- or NP(25)-BSA (high or low affinity, respectively) (Biosearch Technology), followed by incubation with 1:1000 diluted serum samples, and developed with alkaline phosphatase-conjugated goat anti-mouse IgM, IgG1 or IgG3 (Southern Biotech). Total IgM and IgG were measured in serum samples diluted 1:5000. For IgM, the capture antibody was goat anti-IgM μ chain-specific (Millipore AP500) and the conjugated antibody was goat anti-mouse IgM-AP (Southern Biotech., 1020–04). For IgG, the capture antibody was goat anti-IgG (Accurate Chem., SBA 103001) and the conjugated antibody was goat anti-mouse IgG-AP (Sigma, AP308A). Total IgM was measured in culture supernatants diluted 1:100, in IgG in supernatants diluted 1:10. All samples were run in duplicates.

In vitro B cell activation and ELISPOT

B cells were purified by negative selection with magnetic beads (Miltenyi) from the spleen of 4–5 months old anti-dsDNA positive TC and age-matched B6 mice. Cells were cultured in complete RPMI (12.5 mM glucose, 2 mM L-glutamine) at a density of 1 × 106 cells/well in 48-well plates. Two stimulation conditions were tested: “TD”: 10 ug/ml F(ab’)2 anti-IgM (Jackson Immunoresearch Laboratory), 5 ug/ml anti-CD40 mAb (FGK4.5, Bio X-cell) and 25 ng/ml recombinant mouse IL-4 (Peprotech); and “TI”: 1 ug/ml LPS (Sigma). The inhibitors used were 2DG (100 uM), DON (3 uM), BPTES (bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide, 10 nM), 3PO (3-(3-Pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), 10 uM) or medium control. Cells from each mouse were assayed simultaneously in all conditions. Metabolic measurements were conducted after a 16 h culture. Cell viability and proliferation were evaluated with a Cellometer Auto 1000 (Nexcelom) based on the counts of trypan-blue excluding cells. Total IgG and IgM production was evaluated after 4 d of stimulation by ELISPOT as previously described on a Biosys Bioreader 7000-E (18). Prior the assay, serial dilutions of cells were seeded on an ELISPOT plate and cultured overnight with the same stimulation/control medium. Results were calculated as AFC number / 106 cells.

Metabolic measurements

Splenic B cells were purified by negative selection. The Extracellular Acidification Rate (ECAR) and Oxygen Consumption Rate (OCR) were measured using a XF96 Extracellular Flux Analyzer (Agilent Seahorse) in a mitochondrial stress assay with nonbuffered RPMI medium (Sigma) supplemented with 2.5 uM dextrose, 2 mM glutamine and 1 uM sodium pyruvate. Samples were assayed at least in triplicates for 3 successive 8 min time intervals between inhibitor injections. Basal ECAR and OCR values were averaged between replicates for these 3 time points. Spare respiratory capacity (SRC) was defined as the OCR difference between basal and maximum respiration after injection of Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP). Glycolytic capacity (GlC) was calculated as the difference between maximal (after the addition of FCCP) and basal ECAR. Glycolytic and mitochondrial ATP production were calculated according to Agilent Seahorse XFp Real-Time ATP Rate Assay protocol based on proton efflux rate (PER) and OCR values. Glycolysis was also measured in a glycolysis stress test according to the manufacturer’s formula.

Statistical analysis

Differences between groups were evaluated by two-tailed statistics: unpaired or paired t tests with multiple test corrections when applicable using GraphPad Prism v9. Unless specified, graphs show means and standard error of the mean. *P < 0.05, **P < 0.01, and ***P < 0.001.

Results

Lupus-prone TC mice present an elevated B cell metabolism

As previously reported (19), TC mice showed a decreased frequency of follicular (FO) B cells and an increased frequency of marginal zone (MZ) B cells and B-1 cells as compared to B6 control at the pre-autoimmune (Fig. S1A) and autoimmune (Fig. 1A–B) stages. To test whether the metabolism of TC B cells was elevated as compared to B6 controls, we measured OCR (Fig. 1C–E), which corresponds to OXPHOS, and ECAR (Fig. 1F–H), which is primarily attributed to glycolysis. Both basal OCR and ECAR were higher in the B cells from autoimmune TC mice (Fig. 1D and G), which also showed a higher SRC and GlC (Fig. 1E and H). A higher basal OCR and ECAR as well as a higher GlC were also observed in B cells from pre-autoimmune TC mice as compared to their B6 counterparts (Fig. S1B–G). As another measure of metabolism, we evaluated mTOR activation in B cell subsets with p4EBP1 and pS6, which correspond to mTORC1 and pSer473AKT, which corresponds to mTORC2. As previously reported (6; 20), GC B cells presented a higher level of mTORC1 activation as compared to the other subsets (Fig. 1I). More interestingly, all B cell subsets from TC mice presented an increased mTORC1 activation, especially with p4EBP1 expression (Fig. 1I). pAKT levels were similar between TC and B6 B cells, except for TC GC B cells in which it was higher than in B6. Overall, these results show that B cells from TC mice present an elevated OXPHOS and glycolysis as well as mTORC1 activation that precede overt manifestations of autoimmunity, as we have previously reported for CD4+ T cells (13; 16).

Figure 1. B cells from TC mice show an enhanced metabolism.

Figure 1.

Open in a new tab

Representative FACS plots (A) and frequency (B) of FO B (IgM+/LoCD23+), MZ B (IgM+CD23−CD21HiCD1dHi), and B-1 cells (IgM+CD23−CD21LoCD1dLo) in mature B cells (B220+CD93−). C-H. Mitochondrial stress test conducted on total B cells. OCR time-course (C), basal level (D) and SRC (E). ECAR time-course (F), basal (G) and GlC (H). I. mTOR activation measured by the mean fluorescence intensity (MFI) of pE4BP1, pS6 and pAKT expression in the subsets shown in (A) as well as in GC B cells. B cells were obtained from the spleen of 7 – 10 months old B6 and TC mice. N = 5 (B), N = 7 – 9 (C-H) and N = 5 – 9 (I) per group. t tests, with Dunnett’s multiple test corrections (I), *P < 0.05, **P < 0.01, and ***P < 0.001.

The metabolism of TC B cells at steady state is fueled by glucose

We confirmed that the treatment of anti-dsDNA IgG-positive TC mice with 2DG reduced the frequency and absolute number of TFH and GC B cells as well as plasma cells to levels similar to that of treated age-matched B6 controls (Fig. S2A–D) (16). 2DG-treated TC mice showed a slightly decreased frequency of total B cells and B-1 cells, but no change in FO and MZ B cells (Fig. 2A and Fig. S2E–F). This suggests that glycolysis is required for the spontaneous development of GC B and plasma cells, but not to maintain other B cell developmental stages. The inhibition of glycolysis had no effect on B6 B cells, although a trend for a decreased frequency of B-1 cells was also observed. 2DG decreased respiration and glycolysis in TC B cells to B6 levels (Fig. 2B–G), indicating that the increased metabolism in TC B cells was largely driven by glucose. Although SRC was not affected, 2DG expectedly decreased GlC in both strains (Fig. 2D and G). 2DG reduced mTORC1 and mTORC2 activation in TC GC B cells (Fig. 2H), the subset in which mTOR activation was the highest (Fig. 1E). 2DG also reduced mTORC1 activation in MZB and B-1 cells, but had no effect on FO B cells (Fig.2H).

Figure 2. 2DG treatment normalizes the metabolism of B cells in TC mice.

Figure 2.

Open in a new tab

B6 and TC mice were treated with 2DG for 4 weeks starting at 5 – 7 months of age and compared to age-matched controls. A. Frequency of splenic FO B, MZ B, and B-1 cells. B-G. Mitochondrial stress test conducted on total B cells. OCR time-course (B), basal level (C), and SRC (D). ECAR time-course (E), basal level (F) and GlC (G). H. MFI of pE4BP1, pS6 and pAKT expression in the subsets shown in (A) as well as in GC B cells from 2DG-treated or control TC mice. N = 4 – 9 per group. t tests, with Dunnett’s multiple test corrections (H), *P < 0.05, **P < 0.01, and ***P < 0.001.

As previously reported in autoimmune TC mice (16), DON reduced the frequency and number of GC B cells in pre-autoimmune TC mice (Fig. S3A), but it did not affect their plasma cells and TFH cells (Fig. S3B–D). The inhibition of glutaminolysis slightly reduced the frequency of total B cells but had no effect on the frequency and number of non-GC B cell populations (Fig. 3A and Fig. S3E–F). DON treatment did not affect B cell OXPHOS or glycolysis in either strain (Fig. 3B–G). In addition, DON had no effect on mTOR activation in any B cell subset in TC mice (Fig. 3H). These results suggest that, as for CD4+ T cells (16), the elevated metabolism in TC B cells is driven by glucose, with little contribution from glutaminolysis, except for GC B cells.

Figure 3. DON treatment had a minimal effect on B cell metabolism in TC mice.

Figure 3.

Open in a new tab

B6 and TC mice were treated with DON for 2 weeks starting at 5 – 7 months of age and compared to age-matched controls. A. Frequency of FO B, MZ B, and B-1 cells. B-G. Mitochondrial stress test conducted on total B cells. OCR time-course (B), basal level (C), and SRC (D). ECAR time-course (E), basal level (F) and GlC (G). H. MFI of pE4BP1, pS6 and pAKT expression in the subsets shown in (A) as well as in GC B cells from TC mice treated with DON or controls. N = 4 – 5 mice per group. t tests, *P < 0.05 and **P < 0.01.

Glutaminolysis but not glycolysis is required by the TD humoral response

We have previously reported that the humoral response to secondary TD immunization was inhibited by DON but not by 2DG, while autoantibody production was inhibited by both (16). Here, we further analyzed how the inhibition of glycolysis or glutaminolysis affects B cells and their metabolism during a primary immune response to the TD exogenous antigen, NP-KLH (Fig. 4). In addition, we assessed the role of mTOR activation by treating immunized mice with RAPA. TD immunization eliminated the strain differences in B cell basal metabolism, but B cells from TD-immunized TC mice maintained higher respiratory and glycolytic reserves than B6 counterparts (Fig. 4A and E–G). 2DG increased respiration, glycolysis, and both mitochondrial and glycolytic ATP production in total B cells from TD-immunized B6 mice, but DON had no effect, except a small increase in SRC (Fig. 4A–H). The metabolism of B cells from TD-immunized TC mice was little affected by either 2DG or DON, but DON decreased their mitoATP production (Fig. 4D).

Figure 4. 2DG and DON differently affect B cell metabolism during a TD-humoral response.

Figure 4.

Open in a new tab

Mitochondrial stress test conducted on total B cells from B6 and TC mice treated with 2DG, DON or control, 10 d after immunization with NP-KLH. A. OCR time course in B6 (left) and TC (right) mice. B. Basal OCR. C. SRC. D. ATP production through mitochondrial respiration. E. ECAR time-course in B6 (left) and TC (right) mice. F. Basal ECAR. G. GlC. H. ATP production through glycolysis. N = 4 – 10 mice per group. For simplification, statistical differences between strains are not shown. Dunnett’s multiple comparison tests, *P < 0.05, **P < 0.01, and ***P < 0.001.

As previously reported (16), DON reduced the frequency and number of total GC B cells and plasma cells in both B6 and TC immunized mice, whereas 2DG inhibited the differentiation of GC B cells only in TC mice (Fig. 5A and C). Neither 2DG or DON affected the differentiation of NP-specific GC B cells and plasma cells relative to total GC B cells and plasma cells in B6 mice, but these measures of antigen-specific B cell differentiation were increased by 2DG in TC mice (Fig. 5B and D). These results suggest that glutaminolysis is globally required for the differentiation into GC B cells and plasma cells, and that 2DG may shift the balance between autoreactive and antigen-induced GC B and plasma cells in favor of the latter. Total IgM was reduced in 2DG-treated mice from both strains, and total IgG was increased only in 2DG-treated B6 mice, while DON had no effect on either (Fig. 5E). The production of NP-specific IgM was reduced by DON in both strains but reduced by 2DG only in TC mice (Fig. 5F). NP-specific IgG1 was nearly eliminated by DON in both strains, while 2DG treatment resulted in minimal effects with only a decreased level of low affinity NP-specific IgG1 in TC mice (Fig. 5G). Rapamycin treatment had no effect on the differentiation of GC B cells and plasma cells, as well as the production of NP-specific antibodies (Fig. 5A–D, F–G). This suggested that a high level of mTOR signaling is not required for TD responses. Finally, 2DG and DON treatments increased the frequency of MZ B cells in both B6 and TC mice. However, 2DG decreased the frequency of FO B cells only in TC mice, and DON decreased the frequency of B-1 cells only in B6 mice, although a similar trend was observed in TC mice (Fig. 5H). Overall, these results indicated that B cells require glutaminolysis to mount a TD humoral immune response in both B6 and TC mice. Glucose metabolism also contributes to the differentiation of TC B cells into GC B cells, which may pertain to autoreactive rather than immunization-induced GC B cells.

Figure 5. Effect of 2DG, DON and rapamycin treatments on the TD humoral immune response.

Figure 5.

Open in a new tab

A. Frequency of total GC B cells. B. Frequency NP-specific GC B cells among GC B cells. C. Frequency of total plasma cells in splenocytes. D. Frequency of NP-specific plasma cells in plasma cells. E. Serum total IgM and IgG. F. Serum anti-NP25 IgM. G. Serum high-affinity anti-NP4 and low affinity anti-NP25 IgG1. H. Frequency of FO B, MZ B, and B-1 cells in splenic mature B cells. N = 3 – 13 mice per group. For simplification, statistical differences between strains are not shown. Dunnett’s multiple comparison tests with control groups, *P < 0.05, **P < 0.01, and ***P < 0.001.

The TI-humoral response requires both glycolysis and glutaminolysis

We next evaluated how the inhibition of glycolysis or glutaminolysis affects B cells during the TI response to immunization with NP-LPS. Contrary to the TD response (Fig. 4), 2DG and DON decreased basal respiration and glycolysis (Fig. 6A–B), as well as both types of ATP production (data not shown) in the B cells from TI-immunized B6 mice. 2DG further decreased the respiratory and glycolytic reserves in these B6 B cells. 2DG and DON had however no effect on either basal respiration, glycolysis or ATP production in the B cells from TI-immunized TC mice (Fig. 6A–B). B-1 and MZ B cells are the subsets largely responsible for TI–antibody production (21). DON increased the frequency of FO B cells, but it decreased the frequency of B-1 cells in both immunized B6 and TC mice (Fig. 6C). In addition, DON decreased the frequency of MZ B cells in B6 mice and 2DG increased the frequency of B-1 cells in TC mice. These results suggest that splenic B-1 cells are relatively more dependent on glutaminolysis than other subsets in TI conditions. Both 2DG and DON reduced the frequency of total plasma cells in TI-immunized B6 mice, while the frequency of plasma cells was not affected by 2DG in TC mice (Fig. 6D). In addition, 2DG but not DON further decreased the relative frequency of NP-specific plasma cells. Nonetheless, the number of NP-specific plasma cells was greatly reduced by both inhibitors in both strains (Fig. 6D). TI antigens generate short-lived GCs with limited class-switching and affinity maturation (22; 23). Both 2DG and DON greatly reduced the frequency of total GC B cells in both strains (Fig. 6E), which contrasts with the dominant effect of DON on GC B cells in the TD response (Fig. 5A). As for TD immunization, the relative frequency of NP-specific GC B cells was not reduced by either inhibitor. Finally, both 2DG and DON greatly reduced the production of antibodies in both strains, either total IgM or IgG3 (Fig. 6F and G), or NP-specific IgM, IgG3 and IgG1. These results suggest that B cells require both glycolysis and glutaminolysis to produce TI antibodies in both lupus-prone and control strains, although B6 and TC B cells maintain different metabolic programs in these conditions.

Figure 6. Effect of 2DG and DON treatments on the TI humoral immune response.

Figure 6.

Open in a new tab

A-B. Mitochondrial stress test conducted on total splenic B cells 7 d after immunization with NP-LPS. A. Basal OCR and SRC. B. Basal ECAR and GlC. C. Frequency of FO B, MZ B, and B-1 cells in splenic mature B cells. D. Frequency of total and NP-specific plasma cells, as well as number of NP-specific plasma cells. E. Frequency of total GC B cells and NP-specific GC B cells among GC B cells. Serum total IgM (F) and IgG3 (G). G. Serum levels of anti-NP25 IgM, anti-NP25 IgG3 and anti-NP25 IgG1. N = 3 – 9 mice per group. For simplification, statistical differences between strains are not shown. Dunnett’s multiple comparison tests, *P < 0.05, **P < 0.01, and ***P < 0.001.

Glycolysis but not glutaminolysis is required for the TI and TD differentiation of B cells in vitro

To assess the intrinsic metabolic requirements of TC relative to B6 B cells, we used an in vitro assay in which purified B cells were stimulated with a combination of anti-IgM and anti-CD40 antibodies with recombinant IL-4 to represent the TD response, and with LPS to represent the TI response, in the presence of 2DG or 3PO to inhibit glycolysis, and DON or BPTES to inhibit glutaminolysis. These inhibitors did not impair cell viability as compared to untreated cells (data not shown). After 4 d of stimulation, TC B cells differentiated into a larger number of IgM AFCs under TI conditions (Fig. 7A), but there was no difference between strains for IgG AFCs under TD conditions (Fig. 7B). 2DG markedly reduced AFC differentiation in both TI or TD conditions, whereas DON had no effect or even slightly increased TI-induced B6 IgM AFCs and TD-induced TC IgG AFCs (Fig. 7A and B). The reduction of IgM production in TI conditions (Fig. 7C) and IgG production in TD conditions (Fig. 7D) by the inhibition of glycolysis was confirmed with 3PO in an independent cohort. We also confirmed with BPTES that the inhibition of glutaminolysis did not decrease IgM production (Fig. 7D). We also showed that TC B cells imported more glucose than B6 B cells in TI but not in TD conditions, as shown by 2NBDG uptake (Fig. 7E and F). This result corresponds to the greater IgM AFC differentiation by TC B cells in TI conditions. FA uptake, which has been shown to promote plasma cell differentiation (24), was higher in TC than B6 B cells in both conditions (Fig. 7C and D).

Figure 7. Glycolysis but not glutaminolysis is required by B cells for antibody production in vitro.

Figure 7.

Open in a new tab

B cells purified from 4 – 5 months old B6 and TC mice were cultured in TI or TD conditions in the presence of 2DG, 3PO, DON, BPTES, or in medium control. Differentiation into AFCs as well as glucose and FA uptakes were evaluated after 4 d. Frequency of AFCs producing IgM in TI (A) and IgG in TD (B) conditions. Representative ELISPOT images are shown on the left and the numbers of AFCs per 106 cells are shown separately for 2DG and DON with the control values being the same for both inhibitors, N = 7 – 10. IgM production in TI culture (C) and IgG production in TD culture (D) supernatants, N = 3 – 6. 2NBDG and bodipy uptake in TI (C) and TD (D) conditions, N = 3. Paired t tests between controls and treated cells, unpaired t test between B6 and TC cells. D: one-tailed t tests. *P < 0.05, **P < 0.01, and ***P < 0.001.

We next evaluated whether the differences in AFC differentiation between strains and in response to 2DG treatment corresponded to early changes in respiration and glycolysis. Under TI conditions, TC B cells showed a higher basal respiration and glycolysis, assessed with a mitochondrial stress test, as well as glycolysis assessed with a glycolysis stress test, than B6 B cells (Fig. S4A). These parameters were reduced by 2DG only in TC B cells, which was confirmed with 3PO (data not shown). Under TD conditions, glycolysis assessed by the glycolysis stress test was higher in TC than B6 B cells, and it was also reduced only in TC B cells by 2DG (Fig. S4B) and 3PO (data not shown). However, the basal respiration and glycolysis were similar between strains and not reduced by 2DG. Instead, there was a trend for an increased respiration in 2DG-treated B cells under TD conditions (Fig. S4B). DON decreased the respiration of TC B cells in TI conditions and increased it in B cells from both strains in TD conditions (Fig. S4A and B). Therefore, 2DG and DON exert similar effects on the respiration of B cells, although going in opposite directions in TI and TD conditions. DON also decreased ECAR in TC B cells in TI conditions, just as did 2DG.

Finally, we first assessed mTOR activation in these assays. As observed in vivo (Fig. 1I), p4EBP1 expression was higher in TC than in B6 B cells, and it was slightly decreased by DON in TC B cells in TI conditions and by 2DG in B6 B cells in TD conditions (Fig. S4C). pS6 expression was similar between TC and B6 B cells and not affected by either inhibitor in TI conditions (Fig. S4D). pS6 expression was lower in TC B cells than in B6 B cells in TD conditions and it was greatly inhibited by either glycolysis and glutaminolysis in B cells from both strains (Fig. S4D). Finally, pAKT expression was also similar between TC and B6 B cells under TI conditions, but with a decrease in response to DON (Fig. S4E). In TD condition, pAKT expression was lower in TC than in B6 B cells, and it was reduced by DON treatment in both B6 and TC B cells and by 2DG only in B6 B cells. These results suggest that the intrinsic mTOR activation in B cells is different in TD and TI conditions, with a greater sensitivity to glycolytic and specially glutaminolysis inhibitors in TD conditions.

Overall, the activation of purified B cells revealed that they have an intrinsic requirement for glucose, but not glutamine, to differentiate into AFCs and produce antibody either in TI or TD conditions. Furthermore, TC B cells have an intrinsically higher glycolysis and functional response to LPS activation.

Discussion

Signals such as TLR ligands and BCR ligation, as well as cytokines such as IL-4, promote glucose uptake in activated B cells (3–7). Glucose is used not only as a fuel source by AFCs but it is also required for the glycosylation of newly synthesized antibodies (10). In addition, activated B cells increase their glutamine uptake through CD98 (26). Glutaminolysis feeds into the TCA cycle as α-ketoglutarate, through which it plays an essential role in B cell survival and proliferation under hypoxia and glucose deficiency (27). Glutamine metabolism also regulates chromatin accessibility (28), which is an important component of the genetic programs controlling B cell activation and differentiation, including in lupus (29). However, it is unclear at which specific stages of B cell differentiation glycolysis and glutaminolysis are required, and whether B cells enriched for autoreactive specificities such as in lupus-prone mice, have the same requirements as non-autoimmune B cells. We have shown through treatments with 2DG or DON that spontaneous GC B cells from lupus mice require both glycolysis and glutaminolysis, while GC B cells induced by immunization require only glutaminolysis (16). These experiments did not however address specifically B cell metabolism. Thus, in the present study, we compared B cell metabolism at steady state as an assessment of spontaneous autoreactive activation, as well as during TD or TI humoral immune response between lupus-prone mice and control mice.

We found that B cells from lupus-prone mice present an elevated glycolysis and mitochondrial respiration that precede the production of autoantibodies. The same result was obtained for CD4+ T cells (13). This indicates that dual activation of the two main energy-producing pathways is a common feature of lupus lymphocytes that is not a consequence of autoimmune activation. In vivo treatment with 2DG revealed that glycolysis is the main source of the increased respiration in TC total B cells, and that, as for CD4+ T cells, 2DG does not affect the metabolism of non-autoimmune B6 B cells. DON treatment showed that glutaminolysis is not required for the elevated respiration in TC B cells and that it does not contribute indirectly to TC B cell glycolysis, which has been shown in Th17 cells (30). We have reported that the inhibition of either glycolysis or glutaminolysis limited the expansion of GC B cells in lupus mice. Here, we showed that this dual requirement does not extend to MZ B and B-1 cells, two other B cell subsets that are expanded in lupus, including the TC model (19; 31). The expansion of TC B-1 cells required glycolysis while the expansion of TC MZ B cells was unchanged by either inhibitor. These results suggest that, at steady state, most B cells in either lupus or control mice do not have a specific requirement for either glucose or glutamine, and highlight the uniqueness of autoreactive GC B cells, which require both.

We found that all TC B cell subsets presented a higher level of mTORC1 activation than B6 B cells at steady state. These results corroborated the higher mTORC1 activation reported in total B cells expressing the Sle1 and Sle3 loci, which are included in TC mice, after a strong activation in vitro (15). 2DG reduced mTOR activation in TC GC B cells, indicating that they have, as TC TFH cells, an increased glucose-dependent mTORC1 activation (16). mTOR activation in TC FO B cells was however not dependent on glycolysis, which may be consistent with a previous report showing that mTORC1 was not required for the induction of glycolysis after BCR stimulation in vitro (3). Glutamine activates mTORC1 through a RAG GTPAse-independent pathway (32), but this direct sensing is independent of glutaminolysis, and therefore it was not tested in this study. Overall, these results highlight the similarity between lupus CD4+ T and B cells, and specifically GC B cells and TFH cells for their dependency on glycolysis and mTORC1 activation. In addition, we showed here that the production of TD-antibodies is not mTOR-dependent. Moreover, while a high level of mTORC1 activation is maintained in purified TC B cells activated in vitro, the observed changes in mTOR activation in response to 2DG or DON do not correlate with the effect of these inhibitors on AFC differentiation. While it has been shown in multiple models of lupus-prone mice that the production of autoantibodies is mTOR-dependent (15; 33; 34), our results suggest that mTOR activation is not a central player in the production of either TI or TD antibodies.

The analysis of the effect of 2DG and DON on the production of TI and TD antibodies in vivo and in vitro revealed similar metabolic requirements between lupus-prone and control mice that were context-dependent (Table 1). The production of IgM induced in vitro or in vivo always depends on glycolysis, while it depends on glutaminolysis only in vivo, suggesting that it is not intrinsic to B cells. As previously shown (16), the production of IgG induced in vivo depends on glutaminolysis while IgG produced by purified B cells depends on glycolysis, Notably, the production of anti-dsDNA IgG in vivo requires both (16). These results suggest an intrinsic requirement of TD-activated B cells for glucose to differentiate into AFCs that is modified to a requirement for glutamine in the presence of T cells and other supporting cells in vivo. It also suggests that autoreactive B cells maintain this intrinsic glucose requirement is maintained in spite of the presence of supporting cells. The role of FA oxidation, which is intrinsically required by B cells to respond to TD immunization in vivo (9), has not been evaluated in the production of autoantibodies.

Table 1.

Summary of the effect of 2DG and DON on antibodies produced in B6 and TC mice

Purified B cells In vivo
TI IgM TD IgG TI IgM TI IgG TD IgM TD IgG αDNA-IgG*
2DG DON 2DG DON 2DG DON 2DG DON 2DG DON 2DG DON 2DG DON
B6 ↓ ↑ ↓ = ↓ ↓ ↓ ↓ ↓ ↓ = ↓ NA
TC ↓ = ↓ ↑ ↓ ↓ ↓ ↓ ↓ ↓ = ↓ ↓ ↓
Open in a new tab
*

Results from (16).

Up and down arrows indicate increased and decreased antibody production with the inhibitor relative to controls. = indicates no change.

Metabolically, B6 and TC B cells responded differently to TD immunization. The inhibition of glycolysis increased respiration in B6 B cells. A similar increased was observed in purified B cells from both strains stimulated under TD conditions. These results are consistent with FAO driving GC B cell differentiation in response to protein immunization (9). On the other hand, respiration was decreased by 2DG in B cells from TI-immunized B6 mice as well as in purified TC B cells stimulated under TI conditions. These metabolic differences are likely to reflect differences in signaling pathways triggered in each condition with TD stimulation favoring FA oxidation and TI stimulation favoring glycolysis. It should be noted that the downregulation of B cell glycolysis by 2DG at steady state in TI-immunized mice, as well as in purified TC B cells corresponds to the inhibitory effect of 2DG on autoantibody production or TI-induced antibody in vivo and in vitro, respectively. On the other hand, the uncoupling between 2DG and B cell glycolysis in TD immunization is associated with a lack of inhibition of 2DG on TD-induced antibodies.

DON treatment had surprisingly little effect on the metabolism of B cells although it showed functional effects on cellular expansion and antibody production in vivo. We confirmed that DON blunted the expansion of GC B cells in both TD-immunized B6 and TC mice, and profoundly inhibited the production of antigen-specific antibodies as well as autoantibodies. The lack of inhibitory effect of DON on antibody production by purified B cells, as well as its lack of effect on B cell metabolism defined as respiration, glycolysis and mTOR activation, suggest that glutaminolysis may regulate B cells through epigenetic modification, most likely, indirectly through the metabolism and/or epigenetics of non-B cells, probably CD4+ T cells. Finally, while T cells provides the optimal help for class-switching, some TI-responses also produce short-lived GCs and class-switched antibodies induced by a variety of signals (35–38). Our result showed that glutaminolysis is the main energy source for TD-GCs and class-switching, whereas TI-GC and class-switching utilized both glucose and glutamine.

It should be noted that the metabolic responses of in vitro activated purified B cells to 2DG and DON assessed by extracellular flux assays and mTOR activation did not completely match with the effect of these inhibitors on their AFC differentiation and antibody production. While the latter represents a functional effect, the metabolic parameters may be skewed by the culture medium, which is likely to have supraphysiological levels of nutrients, including glucose and glutamine, making them potentially less reliable.

Overall this study revealed that the requirements of B cells for glycolysis and glutaminolysis are subset- and condition-dependent and that they differ between lupus and non-autoimmune mice. Glucose dependency of B cells was more stringent in the absence of T cells, which may provide survival signals that bypass the need for the glucose / PI3K axis. The high requirements of autoreactive GC B cells and ensuing producing of class-switch autoantibodies for glucose may be related to intrinsically higher levels of signaling in lupus B cells (15), or may be a secondary consequence of the glucose requirements of autoreactive TFH cells. A limitation of our study was that the results were obtained with pharmacological interventions, which may or may not accurately reflect the function of true “glycolysis” or “glutaminolysis” in B cells. Follow-up studies should be performed with cell-specific genetic targeting of these pathways. In addition, metabolic analyses using extra-cellular flow assays are not equipped to evaluate ex vivo polyclonal B cell subsets, and the results obtained with total B cells do not account for differences that are likely to exist between B cell subsets, and that may have significant functional consequences. New technological advances, especially scRNASeq platforms focusing on the expression of genes controlling metabolic networks (39; 40) are likely to change the field in the near future.

Supplementary Material

1

Key Points:

  • Lupus B cells present an elevated metabolism preceding the onset of autoimmunity.

  • Except for GC B cells, glucose largely drives the metabolism of lupus B cells.

  • Lupus and control B cells require glycolysis and glutaminolysis in TI-responses.

Acknowledgments.

We thank the Morel lab members for technical assistance and discussion.

This work was supported by a grant from the National Institutes of Health R01 AI128901 to LM.

References

  • 1.Goodnow CC, Vinuesa CG, Randall KL, Mackay F, and Brink R. 2010. Control systems and decision making for antibody production. Nat Immunol. 11:681–688. [DOI] [PubMed] [Google Scholar]
  • 2.Choi SC, and Morel L. 2020. Immune metabolism regulation of the germinal center response. Exp Mol Med. 52:348–355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Doughty CA, Bleiman BF, Wagner DJ, Dufort FJ, Mataraza JM, Roberts MF, and Chiles TC. 2006. Antigen receptor–mediated changes in glucose metabolism in B lymphocytes: role of phosphatidylinositol 3-kinase signaling in the glycolytic control of growth. Blood. 107:4458–4465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Dufort FJ, Bleiman BF, Gumina MR, Blair D, Wagner DJ, Roberts MF, Abu-Amer Y, and Chiles TC. 2007. Cutting Edge: IL-4-mediated protection of primary B lymphocytes from apoptosis via stat6-dependent regulation of glycolytic metabolism. J Immunol. 179:4953–4957. [DOI] [PubMed] [Google Scholar]
  • 5.Caro-Maldonado A, Wang R, Nichols AG, Kuraoka M, Milasta S, Sun LD, Gavin AL, Abel ED, Kelsoe G, Green DR, and Rathmell JC. 2014. Metabolic reprogramming is required for antibody production that is suppressed in anergic but exaggerated in chronically BAFF-exposed B cells. J Immunol. 192:3626–3636. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Ersching J, Efeyan A, Mesin L, Jacobsen JT, Pasqual G, Grabiner BC, Dominguez-Sola D, Sabatini DM, and Victora GD. 2017. Germinal center selection and affinity maturation require dynamic regulation of mTORC1 kinase. Immunity. 46:1045–1058.e1046. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Jellusova J, Cato MH, Apgar JR, Ramezani-Rad P, Leung CR, Chen C, Richardson AD, Conner EM, Benschop RJ, Woodgett JR, and Rickert RC. 2017. Gsk3 is a metabolic checkpoint regulator in B cells. Nat Immunol. 18:303–312. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Jayachandran N, Mejia EM, Sheikholeslami K, Sher AA, Hou S, Hatch GM, and Marshall AJ. 2018. TAPP adaptors control B cell metabolism by modulating the phosphatidylinositol 3-kinase signaling pathway: A novel regulatory circuit preventing autoimmunity. J Immunol. 201:406–416. [DOI] [PubMed] [Google Scholar]
  • 9.Weisel FJ, Mullett SJ, Elsner RA, Menk AV, Trivedi N, Luo W, Wikenheiser D, Hawse WF, Chikina M, Smita S, Conter LJ, Joachim SM, Wendell SG, Jurczak MJ, Winkler TH, Delgoffe GM, and Shlomchik MJ. 2020. Germinal center B cells selectively oxidize fatty acids for energy while conducting minimal glycolysis. Nat Immunol. 21:331–342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Lam WY, Becker AM, Kennerly KM, Wong R, Curtis JD, Llufrio EM, McCommis KS, Fahrmann J, Pizzato HA, Nunley RM, Lee J, Wolfgang MJ, Patti GJ, Finck BN, Pearce EL, and Bhattacharya D. 2016. Mitochondrial pyruvate import promotes long-term survival of antibody-secreting plasma cells. Immunity. 45:60–73. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Vinuesa CG, Sanz I, and Cook MC. 2009. Dysregulation of germinal centres in autoimmune disease. Nat Rev Immunol. 9:845–857. [DOI] [PubMed] [Google Scholar]
  • 12.Elsner RA, and Shlomchik MJ. 2020. Germinal center and extrafollicular B cell responses in vaccination, immunity, and autoimmunity. Immunity. 53:1136–1150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Yin Y, Choi SC, Xu Z, Perry DJ, Seay H, Croker BP, Sobel ES, Brusko TM, and Morel L. 2015. Normalization of CD4+ T cell metabolism reverses lupus. Sci Transl Med. 7:274ra218. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Yin Y, Choi S-C, Xu Z, Zeumer L, Kanda N, Croker BP, and Morel L. 2016. Glucose oxidation is critical for CD4+ T cell activation in a mouse model of systemic lupus erythematosus. J Immunol. 196:80–90. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Wu T, Qin X, Kurepa Z, Kumar KR, Liu K, Kanta H, Zhou XJ, Satterthwaite AB, Davis LS, and Mohan C. 2007. Shared signaling networks active in B cells isolated from genetically distinct mouse models of lupus. J Clin Invest. 117:2186–2196. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Choi SC, Titov AA, Abboud G, Seay HR, Brusko TM, Roopenian DC, Salek-Ardakani S, and Morel L. 2018. Inhibition of glucose metabolism selectively targets autoreactive follicular helper T cells. Nat Commun. 9:4369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Morel L, Croker BP, Blenman KR, Mohan C, Huang G, Gilkeson G, and Wakeland EK. 2000. Genetic reconstitution of systemic lupus erythematosus immunopathology with polycongenic murine strains. Proc Natl Acad Sci USA. 97:6670–6675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Sang A, Niu H, Cullen J, Choi SC, Zheng YY, Wang H, Shlomchik MJ, and Morel L. 2014. Activation of rheumatoid factor-specific B cells is antigen dependent and occurs preferentially outside of germinal centers in the lupus-prone NZM2410 mouse model. J Immunol. 193:1609–1621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Duan B, Niu H, Xu Z, Sharpe AH, Croker BP, Sobel ES, and Morel L. 2008. Intrafollicular location of marginal zone / CD1d hi B cells is associated with autoimmune pathology in a mouse model of lupus. Lab Invest. 88:1008–1019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Raybuck AL, Cho SH, Li J, Rogers MC, Lee K, Williams CL, Shlomchik M, Thomas JW, Chen J, Williams JV, and Boothby MR. 2018. B cell-intrinsic mTORC1 promotes germinal center-defining transcription factor gene expression, somatic hypermutation, and memory B cell generation in humoral immunity. J Immunol. 200:2627–2639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Fagarasan S, and Honjo T. 2000. T-Independent immune response: new aspects of B cell biology. Science. 290:89–92. [DOI] [PubMed] [Google Scholar]
  • 22.de Vinuesa CG, Cook MC, Ball J, Drew M, Sunners Y, Cascalho M, Wabl M, Klaus GG, and MacLennan IC. 2000. Germinal centers without T cells. J Exp Med. 191:485–494. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Lentz VM, and Manser T. 2001. Cutting edge: germinal centers can be induced in the absence of T cells. J Immunol. 167:15–20. [DOI] [PubMed] [Google Scholar]
  • 24.Kobayashi S, Phung HT, Tayama S, Kagawa Y, Miyazaki H, Yamamoto Y, Maruyama T, Ishii N, and Owada Y. 2021. Fatty acid-binding protein 3 regulates differentiation of IgM-producing plasma cells. Febs j. 288:1130–1141. [DOI] [PubMed] [Google Scholar]
  • 25.Sasawatari S, Okamoto Y, Kumanogoh A, and Toyofuku T. 2020. Blockade of N-glycosylation promotes antitumor immune response of T cells. J Immunol. 204:1373–1385. [DOI] [PubMed] [Google Scholar]
  • 26.Cantor J, Browne CD, Ruppert R, Feral CC, Fassler R, Rickert RC, and Ginsberg MH. 2009. CD98hc facilitates B cell proliferation and adaptive humoral immunity. Nat Immunol. 10:412–419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Le A, Lane AN, Hamaker M, Bose S, Gouw A, Barbi J, Tsukamoto T, Rojas CJ, Slusher BS, Zhang H, Zimmerman LJ, Liebler DC, Slebos RJ, Lorkiewicz PK, Higashi RM, Fan TW, and Dang CV. 2012. Glucose-independent glutamine metabolism via TCA cycling for proliferation and survival in B cells. Cell Metab. 15:110–121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Yoo HC, Yu YC, Sung Y, and Han JM. 2020. Glutamine reliance in cell metabolism. Exp Mol Med. 52:1496–1516. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Bacalao MA, and Satterthwaite AB. 2020. Recent advances in lupus B cell biology: PI3K, IFNγ, and chromatin. Front Immunol. 11:615673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Kono M, Yoshida N, Maeda K, Suarez-Fueyo A, Kyttaris VC, and Tsokos GC. 2019. Glutaminase 1 Inhibition Reduces Glycolysis and Ameliorates Lupus-like Disease in MRL/lpr Mice and Experimental Autoimmune Encephalomyelitis. Arthritis Rheumatol. 71:1869–1878. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Duan B, and Morel L. 2006. Role of B-1a cells in autoimmunity. Autoimmun Rev. 5:403–408. [DOI] [PubMed] [Google Scholar]
  • 32.Jewell JL, Kim YC, Russell RC, Yu FX, Park HW, Plouffe SW, Tagliabracci VS, and Guan KL. 2015. Metabolism. Differential regulation of mTORC1 by leucine and glutamine. Science. 347:194–198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Alperovich G, Rama I, Lloberas N, Franquesa M, Poveda R, Gomà M, Herrero-Fresneda I, Cruzado JM, Bolaños N, Carrera M, Grinyó JM, and Torras J. 2007. New immunosuppresor strategies in the treatment of murine lupus nephritis. Lupus. 16:18–24. [DOI] [PubMed] [Google Scholar]
  • 34.Lui SL, Yung S, Tsang R, Zhang F, Chan KW, Tam S, and Chan TM. 2008. Rapamycin prevents the development of nephritis in lupus-prone NZB/W F1 mice. Lupus. 17:305–313. [DOI] [PubMed] [Google Scholar]
  • 35.Snapper CM, McIntyre TM, Mandler R, Pecanha LM, Finkelman FD, Lees A, and Mond JJ. 1992. Induction of IgG3 secretion by interferon gamma: a model for T cell-independent class switching in response to T cell-independent type 2 antigens. J Exp Med. 175:1367–1371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Gerth AJ, Lin L, and Peng SL. 2003. T-bet regulates T-independent IgG2a class switching. Int Immunol. 15:937–944. [DOI] [PubMed] [Google Scholar]
  • 37.Ueda Y, Liao D, Yang K, Patel A, and Kelsoe G. 2007. T-independent activation-induced cytidine deaminase expression, class-switch recombination, and antibody production by immature/transitional 1 B cells. J Immunol. 178:3593–3601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Pone EJ, Zhang J, Mai T, White CA, Li G, Sakakura JK, Patel PJ, Al-Qahtani A, Zan H, Xu Z, and Casali P. 2012. BCR-signalling synergizes with TLR-signalling for induction of AID and immunoglobulin class-switching through the non-canonical NF-κB pathway. Nat Commun. 3:767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Chen D, Wang Y, Manakkat Vijay GK, Fu S, Nash CW, Xu D, He D, Salomonis N, Singh H, and Xu H. 2021. Coupled analysis of transcriptome and BCR mutations reveals role of OXPHOS in affinity maturation. Nat Immunol. 22:904–913. [DOI] [PubMed] [Google Scholar]
  • 40.Wagner A, Wang C, Fessler J, DeTomaso D, Avila-Pacheco J, Kaminski J, Zaghouani S, Christian E, Thakore P, Schellhaass B, Akama-Garren E, Pierce K, Singh V, Ron-Harel N, Douglas VP, Bod L, Schnell A, Puleston D, Sobel RA, Haigis M, Pearce EL, Soleimani M, Clish C, Regev A, Kuchroo VK, and Yosef N. 2021. Metabolic modeling of single Th17 cells reveals regulators of autoimmunity. Cell. 184:4168–4185 e4121. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS1782669-supplement-1.pdf (598.9KB, pdf)

RESOURCES