MetaboLightsapplication/xmlftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14634/m_MTBLS14634_LC-MS_negative_hydrophilic-interaction-liquid-chromatography_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14634/i_Investigation.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14634/s_MTBLS14634.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14634/a_MTBLS14634_LC-MS_negative_hydrophilic-interaction-liquid-chromatography.txtprimary200OKftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14634<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 (79). 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: Conditioned Media.John KoomenMoffitt Cancer CenterT-Lymphocytemass spectrometry assay<p>The metabolites present in 20 μl of the cell culture medium were then extracted using 80 μl of ice-cold MeOH. Following a 30 min incubation on ice and subsequent centrifugation (17,000 x g, 20 min, 4 °C), the supernatant was subjected to LC-HRMS analysis.</p>Mus musculushttps://www.ebi.ac.uk/metabolights/MTBLS14634Marco Davila. Roswell Park Comprehensive Cancer Center. Roswell Park Cancer Center 665 Elm St, Buffalo, NY 14203. marco.davila@roswellpark.org.John Koomen. Moffitt Cancer Center. john.koomen@moffitt.org.<p>Raw data files were converted into cdf files using XCalibur file converter (Thermo).&nbsp;For global metabolomic profiling, peak areas of metabolites were normalized by the median value of identified metabolite peak areas. For data upload to MetaboLights, rawconverter.exe was used to create mzXML files from Thermo .raw data.</p>L-NILCo-cultureINOS knockdownjohn.koomen@moffitt.org<p>For global metabolomics analysis of conditioned cell culture media, the cell-free media were obtained by rapid centrifugation (17,000 x g, 10 sec, room temperature) to collect the supernatant.&nbsp;Conditioned media are compared for CAR-T cells without macrophages, with unpolarized macrophages (unMac), and with immunoregulatory macrophages (imMac).&nbsp;Both iNOS knockout and L-NIL inhibitor treatment were also examined. &nbsp;</p>MetabolomicsThermo Scientific Vanquish UHPLC SystemMetabolomicsMus musculusLC-MSCAR-Tuntargeted analysisT-LymphocyteNitric Oxide SynthaseDiffuse large B cell lymphomaexperimental blankMacrophagesQ Exactive HFThermo Scientific Vanquish UHPLC SystemMetabolomicsMus musculusLC-MSCAR-Tuntargeted analysisT-LymphocyteNitric Oxide SynthaseDiffuse large B cell lymphomaexperimental blankQ Exactive HFMacrophages<p>The 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 lymphomaChimeric 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.2026-05-312026-05-31MTBLS14634