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ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS4647/m_MTBLS4647_LC-MS___metabolite_profiling-1_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS4647/i_Investigation.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS4647/s_MTBLS4647.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS4647/a_MTBLS4647_LC-MS___metabolite_profiling-1.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS4647/FILES/RAW_FILES/20211012_Pool-QC_2_B.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS4647/FILES/RAW_FILES/20211012_E.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS4647/FILES/RAW_FILES/20211012_Blank_3.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS4647/FILES/RAW_FILES/20211012_Pool-QC_3_B.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS4647/FILES/RAW_FILES/20211012_H.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS4647/FILES/RAW_FILES/20211012_Pool-QC_2_A.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS4647/FILES/RAW_FILES/20211012_F.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS4647/FILES/RAW_FILES/20211012_Pool-QC_1_A.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS4647/FILES/RAW_FILES/20211012_Pool-QC_3_A.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS4647/FILES/RAW_FILES/20211012_Pool-QC_1_B.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS4647/FILES/RAW_FILES/20211012_Blank_2.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS4647/FILES/RAW_FILES/20211012_A.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS4647/FILES/RAW_FILES/20211012_C.rawprimary200OKftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS4647Data was normalized versus cell count and log transferred for statistical analysis.Metabolites were identified and further data analysis performed using compound discoverer (version 3.1; ThermoFisher).Metabolite identification with MS1 and MS2 data was performed using mzcloud and mzvault. Further annotations (only MS1) were obtained using ChemSpider, Predicted Composition and Metabolika Search.MetaboLightsPublicLiquid Chromatography MS -The samples were analysed using an ultra high performance liquid chromatograph (Ultimate 3000, Thermo Fisher Scientific) hyphenated to a high resolution mass spectrometer (QExactive Orbitrap Focus, Thermo Fisher Scientific - see next section). Samples were analyzed in reverse phase (RP) chromatography using Acquity UPLC BEH C18 column (1.7 µm, 2.1 x 100 mm, Waters). Chromatographic separation was achieved using a gradient elution, whereby eluent A consisted of water with 0.1% (vol/vol) FA and eluent B of MeOH with 0.1% (vol/vol) FA. The gradient program started with 0.5% B. After injection, the percentage of B increased to 98% in 11 min. This concentration was maintained until 15 min. After 15 min, the concentration of B was reduced to 0.5% (starting conditions) over 0.5 min and then conditioned until 20 min. The flow rate was 0.35 mL/min, the column heater was maintained at 50°C and the autosampler temperature was 4°C. The injection volume was 2 µL.Mitochondrial respiration in B lymphocytes is essential for humoral immunity by controlling flux of the TCA cycle.Friedrich-Alexander-University of Erlangen-NurembergRegina Verena TaudteblankB-lymphocytemass spectrometry assayCells were lysed using 80% MeOH. After centrifugation, supernatants were transferred into HPLC vials, dried and reconstituted in 200 µL eluent.Mus musculusblankhttps://www.ebi.ac.uk/metabolights/MTBLS4647Verena Taudte. verena.taudte@uni-marburg.de.Dirk Mielenz. Friedrich-Alexander-Universität Erlangen-Nürnberg. dirk.mielenz@uk-erlangen.de.No data transformation was performed in this study.ReplicateGenotypeCell countverena.taudte@uni-marburg.deAll experimental procedures were done in agreement with animal protocols approved by the Government of Lower Franconia, Bavaria, Germany. Both female and male mice were used in the experiments. Mice were maintained on a 12-h light/dark cycle with free access to food and water according to governmental rules. K320E-TWINKLE floxed mice[1] were crossed to CD23 CRE mice[2] kindly provided by Meinrad Busslinger) to generate DNT animals. DNT mice used in these experiments had the genetic background DNT+/- CRE+/- and CRE control mice were DNT-/- CRE+/-. The WT animals used in this study were DNT-/- CRE-/- littermates. All mice were on the C57Bl/6 background. Spleen was transferred in cold 2% FCS (in PBS) and gently passed through a 70µm cell strainer (BD) using the plunger of a 5ml syringe (BD). Femur and tibia were flushed with cold 2% FCS using a 27G cannula (BD). Cell suspensions were pelleted by centrifugation at 300xg for 5min at 4°C. Erythrocytes were lysed in red blood cell-lysis buffer (150mM NH4Cl, 10mM KHCO3, 100µM EDTA) for 5 min at room temperature. The reaction was stopped by adding cold 2% FCS before centrifugation at 300xg for 5min at 4 °C. The final cell suspensions were kept in cold 2% FCS after filtration through 30µm mesh filter (Sysmex) B cells were isolated and activated with LPS. 24 h after activation, half of the cells were switched to custom RPE media containing 1g/L U13C glucose (Sigma) for glucose tracing and incubated for another 48 h. Refs:[1] Baris OR, Ederer S, Neuhaus JF, von Kleist-Retzow JC, Wunderlich CM, Pal M, Wunderlich FT, Peeva V, Zsurka G, Kunz WS, Hickethier T, Bunck AC, Stöckigt F, Schrickel JW, Wiesner RJ. Mosaic Deficiency in Mitochondrial Oxidative Metabolism Promotes Cardiac Arrhythmia during Aging. Cell Metab. 2015 May 5;21(5):667-77. doi: 10.1016/j.cmet.2015.04.005. PMID: 25955204[2] Kwon K, Hutter C, Sun Q, Bilic I, Cobaleda C, Malin S, Busslinger M. Instructive role of the transcription factor E2A in early B lymphopoiesis and germinal center B cell development. Immunity. 2008 Jun;28(6):751-62. doi: 10.1016/j.immuni.2008.04.014. PMID: 18538592.Metabolomicsultra-performance liquid chromatography-mass spectrometrypooled quality control sampleMus musculusblankuntargeted analysisHumoral ImmunityMitochondrial DNAMetabolic Flux AnalysisThermo Scientific Dionex Ultimate 3000 HPLC systemexperimental sampletandem mass spectrometryCitrate Cycle (TCA Cycle) PathwayB-lymphocyteuntargeted metabolitesexperimental blankThermo Scientific Q Exactive Focusultra-performance liquid chromatography-mass spectrometrypooled quality control sampleMus musculusblankuntargeted analysisHumoral ImmunityMitochondrial DNAMetabolic Flux AnalysisThermo Scientific Dionex Ultimate 3000 HPLC systemexperimental sampletandem mass spectrometryCitrate Cycle (TCA Cycle) PathwayB-lymphocyteuntargeted metabolitesexperimental blankThermo Scientific Q Exactive FocusThe following Q-Exactive mass spectrometer and HESI settings were maintained for all analyses. Sheath gas, aux gas and sweep gas (all high purity N2) flow rates were set to 60, 20, and 0, respectively. The spray voltage was 4.00 kV. Both, capillary and aux gas heater temperature were set to 400 °C. For MS-analysis a Full MS/dd-MS2 (confirmatory) mode with a resolution of 17.500, a scan-range of 120-1,000 m/z and an automatic gain control target of 1e6  was used. The HESI source was configured in negative mode for ionization.  DL-Malic acidgamma-Aminobutyric AcidTaurineL-PhenylalanineFumaricacid3-Ureidopropionic acidInosinic acid1-Palmitoyl-2-hydroxy-sn-glycero-3-PEL-GlutamineL-TyrosineSuccinyladenosine(R)-2-HydroxyglutarateBeta-AlanineDihydrooroticacidGuanosine5'-monophosphateSuccinic acidL-Aspartic acidUDP-N-acetylglucosamine1-palmitoyl-2-stearoyl-sn-glycero-3-phosphoserineGlutathioneUridine 5'-diphosphogalactosefalseMitochondrial respiration in B lymphocytes is essential for humoral immunity by controlling flux of the TCA cycleTo elucidate the function of oxidative phosphorylation (OxPhos) during B-cell differentiation we employ CD23Cre-driven expression of the dominant-negative K320E mutant of the mitochondrial helicase Twinkle (DNT). DNT-expression depletes mitochondrial DNA during B cell maturation, reduces the abundance of respiratory chain protein subunits encoded by mitochondrial DNA and, consequently, respiratory chain super-complexes in activated B-cells. While B-cell development in DNT-mice is normal, B-cell proliferation, germinal centers and class switch to IgG, plasma cell maturation, T-cell dependent as well as T-cell independent humoral immunity are diminished. DNT expression dampens OxPhos but increases glycolysis in lipopolysaccharide and B-cell receptor activated cells. Lipopolysaccharide activated DNT B-cells exhibit altered metabolites of glycolysis, the pentose phosphate pathway and the tricarboxylic acid cycle, and a lower amount of phosphatidic acid. Consequently, mTORC1 activity and BLIMP1 induction are curtailed while HIF1a is stabilized. Hence, mtDNA controls the metabolism of activated B-cells via OxPhos to foster humoral immunity.2026-08-212022-04-06MTBLS4647MTBLC18307MTBLC16264MTBLC15891MTBLC71169MTBLC15741MTBLC17895MTBLC17295MTBLC18050MTBLC17053MTBLC17202MTBLC58115MTBLC16856MTBLC16865MTBLC18012MTBLC6650MTBLC30865MTBLC16958MTBLC18261MTBLC84520MTBLC176760MTBLC15801CHEBI:18307CHEBI:16264CHEBI:15891CHEBI:71169CHEBI:15741CHEBI:17895CHEBI:17295CHEBI:18050CHEBI:17053CHEBI:17202CHEBI:58115CHEBI:16856CHEBI:16865CHEBI:18012CHEBI:6650CHEBI:30865CHEBI:16958CHEBI:18261CHEBI:84520CHEBI:176760CHEBI:15801