MetaboLightsapplication/xmlftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14640/m_MTBLS14640_LC-MS_negative_hydrophilic-interaction-liquid-chromatography_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14640/i_Investigation.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14640/a_MTBLS14640_LC-MS_negative_hydrophilic-interaction-liquid-chromatography.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14640/s_MTBLS14640.txtprimary200OKftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14640<p>Metabolites were identified by retention time and accurate m/z measurement using an in-house library with EL-MAVEN.</p>MetaboLightsPublicLiquid Chromatography MS - negative - hydrophilic-interaction-liquid-chromatography<p>LC-HRMS analysis was performed on a Vanquish UPLC and a Q-Exactive HF mass spectrometry, employing the same conditions as the previously established methods. A ZIC-pHILIC guard column (4.6 mm ID x 20 mm length, MilliporeSigma, Burlington, MA) and ZIC-pHILIC LC column (4.6 mm ID x 150mm length, 5 μm particle size, MilliporeSigma, Burlington, MA) were used for chromatographic separation at a column temperature of 30 °C. The sample injection volume was 5 μL. The mobile phases consisted of 10 mM (NH4)2CO3 and 0.05% NH4OH in H2O for mobile phase A, and 100% acetonitrile for mobile phase B. The LC gradient conditions were as follows: 0 to 13 min: 80% to 20% of mobile phase B, 13 to 15 min: 20% of mobile phase B.&nbsp;</p>IFN-γ-driven iNOS induction in macrophages mediates CAR T cell resistance in B cell lymphoma (CAR-T Metabolites).John KoomenMoffitt Cancer CenterT-Lymphocytemass spectrometry assay<p>T cells were washed with 1 ml of ice-cold PBS, and metabolites were extracted with 300 μl of 80% methanol via incubation at -80 °C for 15 min. Samples were centrifuged (17,000 x g, 20 min, 4 °C), and supernatants were transferred to an Eppendorf tube and dried in a vacuum evaporator overnight. The dried extracts were resuspended in 20 μl of aqueous 50% methanol, clarified by centrifugation (17,000 x g, 20 min, room temperature), and analyzed by LC-HRMS.</p>Mus musculushttps://www.ebi.ac.uk/metabolights/MTBLS14640Marco Davila. Roswell Park Comprehensive Cancer Center. 665 Elm St, Buffalo, NY 14203, USA. marco.davila@roswellpark.org.John Koomen. Moffitt Cancer Center. 12902 Magnolia Drive Tampa, FL 33612 USA. john.koomen@moffitt.org.<p>Raw data files were converted into cdf files using XCalibur file converter (Thermo). For global metabolomic profiling, peak areas of metabolites were normalized by the median value of identified metabolite peak areas.&nbsp;For data upload to MetaboLights, rawconverter.exe was used to create mzXML files from Thermo .raw data.</p>L-NILCo-culturejohn.koomen@moffitt.org<p>For global metabolomic profiling of CAR T cells comparing the effects of unpolarized macrophages (unMac) or immunoregulatory macrophages (imMac) against no macrophages (NoMac) as well as L-NIL treatment to inhibit iNOS versus vehicle control, 1×10^6 T cells were resuspended in either RPMI-1640 medium (RPMI + 10% heat-inactivated dialyzed FBS). After 4 h incubation, cells were collected and rapidly centrifuged (17,000 x g, 10 sec, room temperature); media was removed.&nbsp;</p>MetabolomicsMetabolomicsVanquishMus musculusLC-MSCAR-Tuntargeted analysisT-LymphocyteNitric Oxide SynthaseDiffuse large B cell lymphomaQ Exactive HFMacrophagesexperimental sampleMetabolomicsMus musculusVanquishLC-MSCAR-Tuntargeted analysisT-LymphocyteNitric Oxide SynthaseDiffuse large B cell lymphomaMacrophagesQ Exactive HFexperimental sample<p>For the Q Exactive HF, ionization was set to negative mode, with the MS scan range set to 60 to 1000 m/z. The mass resolution was 70,000, and the AGC target was 1 x 10^6.&nbsp;</p>falseIFN-γ-driven iNOS induction in macrophages mediates CAR T cell resistance in B cell lymphoma (CAR-T Metabolites)Chimeric antigen receptor (CAR) T cell therapies have revolutionized B cell malignancy treatment, but subsets of patients with large B cell lymphoma (LBCL) experience primary resistance or relapse after CAR T cell treatment. To uncover tumor microenvironment (TME)-induced resistance mechanisms, we examined patients’ intratumoral immune infiltrates and observed that elevated levels of immunoregulatory macrophages in pre-infusion tumor biopsies are correlated with poor clinical responses. In murine models, CAR T cell-produced interferon-gamma (IFN-g) promotes the expression of inducible nitric oxide synthase (iNOS, NOS2) in immunoregulatory macrophages, impairing CAR T cell function. Mechanistically, proteomics analysis of CAR T cells revealed that iNOS-expressing macrophages promote the upregulation of genes mediating apoptosis and cell cycle arrest in CAR T cells, while downregulating ribosome biogenesis and protein synthesis. Furthermore, CAR T cell metabolism is compromised by the depletion of glycolytic intermediates and rewiring of the TCA cycle. Pharmacological inhibition of iNOS enhances the CAR T cell treatment efficacy in B cell tumor-bearing mice. Notably, elevated levels of iNOS+CD14+ monocytes were observed in leukaphereses of patients with non-durable response to CAR T cell therapy. These findings suggest that mitigating iNOS in tumor-associated macrophages (TAMs), potentially by modulating IFN-g expression in CAR T cells, could improve outcomes for LBCL patients.2026-05-312026-05-31MTBLS14640