{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST003265","ANALYSIS_ID":"AN005349","PROJECT_ID":"PR002027","VERSION":"1","CREATED_ON":"June 17, 2024, 8:15 pm"},

"PROJECT":{"PROJECT_TITLE":"Malic Enzyme 2 maintains metabolic state and anti-tumor immunity of CD8+ T cells","PROJECT_SUMMARY":"The functional integrity of CD8+ T cells is closely linked to metabolic reprogramming, therefore, understanding of the metabolic basis of CD8+ T cell activation and antitumor immunity could provide insights into tumor immunotherapy. Here, we report that ME2 is critical for CD8+ T cell activation and immune response against malignancy. ME2 deficiency suppresses CD8+ T cells activation and anti-tumor immune response in vitro and in vivo. Mechanistically, ME2 depletion blocks the TCA cycle flux, leading to the accumulation of fumarate. Fumarate directly binds to DAPK1 and inhibits its activity by competing with ATP for binding. Notably, ME2 deficiency increases the ratio of fumarate to ATP, thereby leading to the inhibition of DAPK1. Consistently, pharmacological inhibition of DAPK1 abolishes the anti-tumor function conferred by ME2 to CD8+ T cells. Collectively, these findings demonstrate a role for ME2 in the regulation of CD8+ T cell metabolism and effector functions, as well as an unexpected function for fumarate as a metabolic signal in the inhibition of DAPK1.","INSTITUTE":"Peking Union Medical College","LAST_NAME":"zhenxi","FIRST_NAME":"zhang","ADDRESS":"Beijing Dongdan Santiao No.5","EMAIL":"13121716766@163.com","PHONE":"010-69156953"},

"STUDY":{"STUDY_TITLE":"Malic Enzyme 2 maintains metabolic state and anti-tumor immunity of CD8+ T cells","STUDY_SUMMARY":"For metabolite analysis, naive ME2+/+ and ME2-/- CD8+ T cells were isolated and activated with anti-CD3 (4μg/ml) and anti-CD28 (1μg/ml). Cells were washed twice with ice-cold PBS and metabolites were extracted with ice-cold 80% methanol. The extracts were analyzed by LC-MS/MS.","INSTITUTE":"Peking Union Medical College","LAST_NAME":"zhang","FIRST_NAME":"zhenxi","ADDRESS":"Beijing Dongdan Santiao No.5","EMAIL":"13121716766@163.com","PHONE":"69156953"},

"SUBJECT":{"SUBJECT_TYPE":"Cultured cells","SUBJECT_SPECIES":"Mus musculus","TAXONOMY_ID":"10090"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"S-1",
"Sample ID":"WT1",
"Factors":{"Genotype":"wild-type","Sample source":"CD8 T cell"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"WT1.RAW"}
},
{
"Subject ID":"S-1",
"Sample ID":"WT2",
"Factors":{"Genotype":"wild-type","Sample source":"CD8 T cell"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"WT2.RAW"}
},
{
"Subject ID":"S-1",
"Sample ID":"WT3",
"Factors":{"Genotype":"wild-type","Sample source":"CD8 T cell"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"WT3.RAW"}
},
{
"Subject ID":"S-1",
"Sample ID":"WT4",
"Factors":{"Genotype":"wild-type","Sample source":"CD8 T cell"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"WT4.RAW"}
},
{
"Subject ID":"S-1",
"Sample ID":"WT5",
"Factors":{"Genotype":"wild-type","Sample source":"CD8 T cell"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"WT5.RAW"}
},
{
"Subject ID":"S-1",
"Sample ID":"KO1",
"Factors":{"Genotype":"Me2-knockout","Sample source":"CD8 T cell"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"KO1.RAW"}
},
{
"Subject ID":"S-1",
"Sample ID":"KO2",
"Factors":{"Genotype":"Me2-knockout","Sample source":"CD8 T cell"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"KO2.RAW"}
},
{
"Subject ID":"S-1",
"Sample ID":"KO3",
"Factors":{"Genotype":"Me2-knockout","Sample source":"CD8 T cell"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"KO3.RAW"}
},
{
"Subject ID":"S-1",
"Sample ID":"KO4",
"Factors":{"Genotype":"Me2-knockout","Sample source":"CD8 T cell"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"KO4.RAW"}
},
{
"Subject ID":"S-1",
"Sample ID":"KO5",
"Factors":{"Genotype":"Me2-knockout","Sample source":"CD8 T cell"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"KO5.RAW"}
},
{
"Subject ID":"S-2",
"Sample ID":"W1",
"Factors":{"Genotype":"wild-type","Sample source":"CD8 T cell"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"W1.RAW"}
},
{
"Subject ID":"S-2",
"Sample ID":"W2",
"Factors":{"Genotype":"wild-type","Sample source":"CD8 T cell"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"W2.RAW"}
},
{
"Subject ID":"S-2",
"Sample ID":"W3",
"Factors":{"Genotype":"wild-type","Sample source":"CD8 T cell"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"W3.RAW"}
},
{
"Subject ID":"S-2",
"Sample ID":"K1",
"Factors":{"Genotype":"Me2-knockout","Sample source":"CD8 T cell"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"K1.RAW"}
},
{
"Subject ID":"S-2",
"Sample ID":"K2",
"Factors":{"Genotype":"Me2-knockout","Sample source":"CD8 T cell"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"K2.RAW"}
},
{
"Subject ID":"S-2",
"Sample ID":"K3",
"Factors":{"Genotype":"Me2-knockout","Sample source":"CD8 T cell"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"K3.RAW"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Mouse CD8+ T cells were isolated from the spleen and lymph nodes of ME2 wild-type and ME2 knockout mice (knockdown of ME2 in T cells). The mice were euthanized and soaked in 75% alcohol for 3 minutes. Separate mouse spleens using a dissector and grind them into single-cell suspensions using a grinder. Single-cell suspension was used for CD8+ T cell isolation using the Mouse Lymphocyte Isolation Kit (Biolegend, 480044, China). T cells were cultured in RMPI-1640 medium supplemented with 10% FBS, 10μg/ml β-mercaptoethanol, and 1% penicillin and streptomycin. For metabolite analysis, CD8+ T cells were stimulated with 4 μg/ml anti-CD3 and 1μg/ml anti-CD28 antibodies for 24 h.","SAMPLE_TYPE":"T-cells","STORAGE_CONDITIONS":"On ice"},

"TREATMENT":{"TREATMENT_SUMMARY":"Naive ME2+/+ and ME2-/- CD8+ T cells were isolated and activated with anti-CD3 (4μg/ml) and anti-CD28 (1μg/ml)."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"For metabolite analysis, naive ME2+/+ and ME2-/- CD8+ T cells were isolated and activated with anti-CD3 (4μg/ml) and anti-CD28 (1μg/ml). Cells were washed twice with ice-cold PBS and metabolites were extracted with ice-cold 80% methanol. The extracts were analyzed by LC-MS/MS.","EXTRACTION_METHOD":"Cells were washed twice with ice-cold PBS and metabolites were extracted with ice-cold 80% methanol.","EXTRACT_STORAGE":"-80℃"},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"HILIC","INSTRUMENT_NAME":"Thermo Dionex Ultimate 3000","COLUMN_NAME":"Merck SeQuant ZIC-HILIC (150 x 2.1mm,5um)","SOLVENT_A":"100% water; 0.1 % formic acid","SOLVENT_B":"100% methanol; 0.1 % formic acid","FLOW_GRADIENT":"0-2 min, 100% phase A; 2-11 min, 0% to 100% B; 11-13 min, 100% B; 13-15 min, 0% to 100% A.","FLOW_RATE":"0.4 ml/min","COLUMN_TEMPERATURE":"35"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive Focus","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"POSITIVE","MS_COMMENTS":"The Q Exactive mass spectrometer was operated in full MS and polarity switching mode. Five independent cell cultures were measured for each condition and samples were randomised in order to avoid bias in sample analyses due to machine drift. XCalibur Qual Browser and XCalibur Quan Browser software (Thermo Scientific), and used for quantification. Experimental samples were randomised in order to avoid bias in sample analyses due to machine drift."},

"MS_METABOLITE_DATA":{
"Units":"Peak area",

"Data":[{"Metabolite":"Adenosine Diphosphate(ADP)","WT1":"5.37E+06","WT2":"3.47E+06","WT3":"3.20E+06","WT4":"3.20E+06","WT5":"2.56E+06","KO1":"2.17E+06","KO2":"3.45E+06","KO3":"3.49E+06","KO4":"2.89E+06","KO5":"2.73E+06","W1":"3566545.25","W2":"2635199.25","W3":"3961358.75","K1":"5360302","K2":"5281175.5","K3":"4863602.5"},{"Metabolite":"Adenosine Monophosphate(AMP)","WT1":"1.15E+06","WT2":"9.14E+05","WT3":"8.25E+05","WT4":"7.20E+05","WT5":"7.22E+05","KO1":"6.95E+05","KO2":"8.96E+05","KO3":"9.10E+05","KO4":"7.79E+05","KO5":"7.87E+05","W1":"923732.81","W2":"674310.56","W3":"544652.69","K1":"443554.91","K2":"612365.56","K3":"376734.59"},{"Metabolite":"Adenosine Triphosphate(ATP)","WT1":"5.09E+07","WT2":"3.07E+07","WT3":"2.84E+07","WT4":"2.96E+07","WT5":"3.43E+07","KO1":"2.39E+07","KO2":"3.60E+07","KO3":"3.19E+07","KO4":"2.32E+07","KO5":"2.04E+07","W1":"654364.06","W2":"738914.56","W3":"690311.44","K1":"1016199.69","K2":"1123779.75","K3":"1393685.12"},{"Metabolite":"Choline","WT1":"9.88E+05","WT2":"1.31E+06","WT3":"1.15E+06","WT4":"1.60E+06","WT5":"2.77E+06","KO1":"2.06E+06","KO2":"1.24E+06","KO3":"1.77E+06","KO4":"1.59E+06","KO5":"1.97E+06","W1":"109157376","W2":"110414752","W3":"108186024","K1":"112415192","K2":"104843560","K3":"122579224"},{"Metabolite":"Citric Acid","WT1":"2.01E+06","WT2":"8.40E+05","WT3":"7.68E+05","WT4":"2.15E+06","WT5":"1.16E+06","KO1":"1.43E+06","KO2":"6.53E+05","KO3":"7.27E+05","KO4":"7.97E+05","KO5":"1.75E+06","W1":"400507.16","W2":"170810.8","W3":"1046639.5","K1":"831950.5","K2":"1175230.38","K3":"2267278"},{"Metabolite":"Creatine","WT1":"1.03E+05","WT2":"1.29E+05","WT3":"1.33E+05","WT4":"2.09E+05","WT5":"2.56E+05","KO1":"2.42E+05","KO2":"1.39E+05","KO3":"1.66E+05","KO4":"2.27E+05","KO5":"2.09E+05","W1":"202860.95","W2":"232698.56","W3":"257040.95","K1":"277272.19","K2":"240844.39","K3":"237221.86"},{"Metabolite":"Fructose 1,6-Bisphosphate","WT1":"4.43E+05","WT2":"2.29E+05","WT3":"1.70E+05","WT4":"2.84E+05","WT5":"1.91E+05","KO1":"1.73E+05","KO2":"1.92E+05","KO3":"1.57E+05","KO4":"1.32E+05","KO5":"1.98E+05","W1":"48961.5","W2":"57595.25","W3":"47305.69","K1":"85321.66","K2":"72985.99","K3":"53750.22"},{"Metabolite":"Fumaric Acid","WT1":"1.09E+05","WT2":"7.04E+04","WT3":"6.77E+04","WT4":"9.97E+04","WT5":"7.33E+04","KO1":"6.50E+05","KO2":"8.11E+05","KO3":"7.36E+05","KO4":"7.53E+05","KO5":"7.12E+05","W1":"3881326","W2":"4596558","W3":"4905779","K1":"40910924","K2":"39579252","K3":"40370740"},{"Metabolite":"Gamma-Aminobutyric Acid","WT1":"1.78E+05","WT2":"2.08E+05","WT3":"1.80E+05","WT4":"2.76E+05","WT5":"4.85E+05","KO1":"3.57E+05","KO2":"1.94E+05","KO3":"2.98E+05","KO4":"2.61E+05","KO5":"3.35E+05","W1":"5985165.5","W2":"6824239.5","W3":"6742473.5","K1":"4787206","K2":"4567318","K3":"5074342"},{"Metabolite":"L-Lactic Acid","WT1":"1.21E+05","WT2":"2.44E+05","WT3":"1.80E+05","WT4":"1.89E+05","WT5":"3.15E+05","KO1":"3.84E+05","KO2":"1.65E+05","KO3":"1.08E+05","KO4":"2.23E+05","KO5":"3.08E+05","W1":"71447112","W2":"92646104","W3":"69461024","K1":"92217152","K2":"65892508","K3":"94951104"},{"Metabolite":"Malic Acid","WT1":"1.83E+06","WT2":"8.10E+05","WT3":"8.17E+05","WT4":"1.33E+06","WT5":"8.88E+05","KO1":"9.81E+06","KO2":"1.18E+07","KO3":"1.07E+07","KO4":"1.05E+07","KO5":"1.03E+07","W1":"30549206","W2":"37206416","W3":"41900972","K1":"334112480","K2":"317340128","K3":"317928064"},{"Metabolite":"NAD","WT1":"2.37E+06","WT2":"1.55E+06","WT3":"1.48E+06","WT4":"2.09E+06","WT5":"1.82E+06","KO1":"1.87E+06","KO2":"2.03E+06","KO3":"1.87E+06","KO4":"1.83E+06","KO5":"2.03E+06","W1":"2667292.25","W2":"2812191.25","W3":"2899324","K1":"3626126.75","K2":"4459451.5","K3":"4102124"},{"Metabolite":"NADH","WT1":"1.80E+05","WT2":"1.46E+05","WT3":"1.37E+05","WT4":"2.03E+05","WT5":"1.08E+05","KO1":"8.80E+04","KO2":"1.04E+05","KO3":"9.15E+04","KO4":"1.00E+05","KO5":"5.78E+04","W1":"389966.59","W2":"474420","W3":"379897.91","K1":"411093.09","K2":"443840.34","K3":"392185.62"},{"Metabolite":"NADP","WT1":"1.57E+06","WT2":"1.06E+06","WT3":"8.62E+05","WT4":"1.20E+06","WT5":"9.25E+05","KO1":"9.45E+05","KO2":"1.32E+06","KO3":"1.05E+06","KO4":"1.01E+06","KO5":"8.71E+05","W1":"0","W2":"3180.16","W3":"94372.81","K1":"0","K2":"0","K3":"110334.02"},{"Metabolite":"NADPH","WT1":"8.34E+04","WT2":"5.84E+03","WT3":"1.01E+05","WT4":"7.78E+03","WT5":"5.53E+04","KO1":"3.61E+04","KO2":"2.55E+04","KO3":"4.23E+04","KO4":"4.60E+03","KO5":"5.44E+03","W1":"40242.52","W2":"49131.62","W3":"11903.74","K1":"53489.03","K2":"112655.94","K3":"33893.9"},{"Metabolite":"Succinic Acid","WT1":"2.90E+06","WT2":"1.81E+06","WT3":"1.73E+06","WT4":"2.33E+06","WT5":"1.91E+06","KO1":"6.03E+06","KO2":"5.99E+06","KO3":"5.64E+06","KO4":"6.18E+06","KO5":"6.89E+06","W1":"6596342.5","W2":"10191412","W3":"7935360","K1":"12713237","K2":"11103603","K3":"13089128"}],

"Metabolites":[{"Metabolite":"Adenosine Diphosphate(ADP)","Formula":"C7H15NO2","KEGG":"C05543","HMDB":"HMDB0006831","Pathway":""},{"Metabolite":"Adenosine Monophosphate(AMP)","Formula":"C3H6O3","KEGG":"C00186","HMDB":"HMDB0000190","Pathway":"Pyruvate Metabolism/Gluconeogenesis"},{"Metabolite":"Adenosine Triphosphate(ATP)","Formula":"C4H9N3O2","KEGG":"C00300","HMDB":"HMDB0000064","Pathway":"Arginine and Proline Metabolism/Glycine and Serine Metabolism"},{"Metabolite":"Choline","Formula":"C20H32N6O12S2","KEGG":"C00127","HMDB":"HMDB0003337","Pathway":"Glutathione Metabolism/Glutamate Metabolism/Arachidonic Acid Metabolism"},{"Metabolite":"Citric Acid","Formula":"C8H20NO6P","KEGG":"C00670","HMDB":"HMDB0000086","Pathway":"Retinol Metabolism"},{"Metabolite":"Creatine","Formula":"C3H9O6P","KEGG":"C00093","HMDB":"HMDB0000126","Pathway":"Glycerolipid Metabolism/Phospholipid Biosynthesis/De Novo Triacylglycerol Biosynthesis/Mitochondrial Electron Transport Chain"},{"Metabolite":"Fructose 1,6-Bisphosphate","Formula":"C5H13NO","KEGG":"C00114","HMDB":"HMDB0000097","Pathway":"Methionine Metabolism/Phospholipid Biosynthesis/Betaine Metabolism"},{"Metabolite":"Fumaric Acid","Formula":"C5H9NO3","KEGG":"C01157","HMDB":"HMDB0000725","Pathway":"Bile Acid Biosynthesis"},{"Metabolite":"Gamma-Aminobutyric Acid","Formula":"C14H20N6O5S","KEGG":"C00021","HMDB":"HMDB0000939","Pathway":"Methionine Metabolism/Folate Metabolism"},{"Metabolite":"L-Lactic Acid","Formula":"","KEGG":"","HMDB":"","Pathway":""},{"Metabolite":"Malic Acid","Formula":"C3H7O7P","KEGG":"C00597","HMDB":"HMDB0000807","Pathway":"Glycolysis"},{"Metabolite":"NAD","Formula":"C5H13O14P3","KEGG":"C00119","HMDB":"HMDB0000280","Pathway":"Purine Metabolism/Pyrimidine Metabolism"},{"Metabolite":"NADH","Formula":"C10H16N5O13P3","KEGG":"C00002","HMDB":"HMDB0000538","Pathway":"Purine Metabolism/Pyrimidine Metabolism/Pentose Phosphate Pathway"},{"Metabolite":"NADP","Formula":"C5H9NO2","KEGG":"C00148","HMDB":"HMDB0000162","Pathway":"Arginine and Proline Metabolism"},{"Metabolite":"NADPH","Formula":"C9H15N3O11P2","KEGG":"C00112","HMDB":"HMDB0001546","Pathway":"Pyrimidine Metabolism"},{"Metabolite":"Succinic Acid","Formula":"C15H23N5O14P2","KEGG":"C00301","HMDB":"HMDB0001178","Pathway":"Nicotinate and Nicotinamide Metabolism/Purine Metabolism/Glutamate Metabolism"}]
}

}