#METABOLOMICS WORKBENCH Zhangzhenxi1995_20240605_222654 DATATRACK_ID:4906 STUDY_ID:ST003265 ANALYSIS_ID:AN005349 PROJECT_ID:PR002027 VERSION 1 CREATED_ON 06-18-2024 #PROJECT PR:PROJECT_TITLE Malic Enzyme 2 maintains metabolic state and anti-tumor immunity of CD8+ T cells PR:PROJECT_SUMMARY The functional integrity of CD8+ T cells is closely linked to metabolic PR:PROJECT_SUMMARY reprogramming, therefore, understanding of the metabolic basis of CD8+ T cell PR:PROJECT_SUMMARY activation and antitumor immunity could provide insights into tumor PR:PROJECT_SUMMARY immunotherapy. Here, we report that ME2 is critical for CD8+ T cell activation PR:PROJECT_SUMMARY and immune response against malignancy. ME2 deficiency suppresses CD8+ T cells PR:PROJECT_SUMMARY activation and anti-tumor immune response in vitro and in vivo. Mechanistically, PR:PROJECT_SUMMARY ME2 depletion blocks the TCA cycle flux, leading to the accumulation of PR:PROJECT_SUMMARY fumarate. Fumarate directly binds to DAPK1 and inhibits its activity by PR:PROJECT_SUMMARY competing with ATP for binding. Notably, ME2 deficiency increases the ratio of PR:PROJECT_SUMMARY fumarate to ATP, thereby leading to the inhibition of DAPK1. Consistently, PR:PROJECT_SUMMARY pharmacological inhibition of DAPK1 abolishes the anti-tumor function conferred PR:PROJECT_SUMMARY by ME2 to CD8+ T cells. Collectively, these findings demonstrate a role for ME2 PR:PROJECT_SUMMARY in the regulation of CD8+ T cell metabolism and effector functions, as well as PR:PROJECT_SUMMARY an unexpected function for fumarate as a metabolic signal in the inhibition of PR:PROJECT_SUMMARY DAPK1. PR:INSTITUTE Peking Union Medical College PR:LAST_NAME zhenxi PR:FIRST_NAME zhang PR:ADDRESS Beijing Dongdan Santiao No.5 PR:EMAIL 13121716766@163.com PR:PHONE 010-69156953 PR:DOI http://dx.doi.org/10.21228/M8M24P #STUDY ST:STUDY_TITLE Malic Enzyme 2 maintains metabolic state and anti-tumor immunity of CD8+ T cells ST:STUDY_SUMMARY For metabolite analysis, naive ME2+/+ and ME2-/- CD8+ T cells were isolated and ST:STUDY_SUMMARY activated with anti-CD3 (4μg/ml) and anti-CD28 (1μg/ml). Cells were washed ST:STUDY_SUMMARY twice with ice-cold PBS and metabolites were extracted with ice-cold 80% ST:STUDY_SUMMARY methanol. The extracts were analyzed by LC-MS/MS. ST:INSTITUTE Peking Union Medical College ST:LAST_NAME zhang ST:FIRST_NAME zhenxi ST:ADDRESS Beijing Dongdan Santiao No.5 ST:EMAIL 13121716766@163.com ST:PHONE 69156953 ST:SUBMIT_DATE 2024-06-05 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS S-2 K1 Genotype:Me2-knockout | Sample source:CD8 T cell RAW_FILE_NAME(Raw file name)=K1.RAW SUBJECT_SAMPLE_FACTORS S-2 K2 Genotype:Me2-knockout | Sample source:CD8 T cell RAW_FILE_NAME(Raw file name)=K2.RAW SUBJECT_SAMPLE_FACTORS S-2 K3 Genotype:Me2-knockout | Sample source:CD8 T cell RAW_FILE_NAME(Raw file name)=K3.RAW SUBJECT_SAMPLE_FACTORS S-1 KO1 Genotype:Me2-knockout | Sample source:CD8 T cell RAW_FILE_NAME(Raw file name)=KO1.RAW SUBJECT_SAMPLE_FACTORS S-1 KO2 Genotype:Me2-knockout | Sample source:CD8 T cell RAW_FILE_NAME(Raw file name)=KO2.RAW SUBJECT_SAMPLE_FACTORS S-1 KO3 Genotype:Me2-knockout | Sample source:CD8 T cell RAW_FILE_NAME(Raw file name)=KO3.RAW SUBJECT_SAMPLE_FACTORS S-1 KO4 Genotype:Me2-knockout | Sample source:CD8 T cell RAW_FILE_NAME(Raw file name)=KO4.RAW SUBJECT_SAMPLE_FACTORS S-1 KO5 Genotype:Me2-knockout | Sample source:CD8 T cell RAW_FILE_NAME(Raw file name)=KO5.RAW SUBJECT_SAMPLE_FACTORS S-2 W1 Genotype:wild-type | Sample source:CD8 T cell RAW_FILE_NAME(Raw file name)=W1.RAW SUBJECT_SAMPLE_FACTORS S-2 W2 Genotype:wild-type | Sample source:CD8 T cell RAW_FILE_NAME(Raw file name)=W2.RAW SUBJECT_SAMPLE_FACTORS S-2 W3 Genotype:wild-type | Sample source:CD8 T cell RAW_FILE_NAME(Raw file name)=W3.RAW SUBJECT_SAMPLE_FACTORS S-1 WT1 Genotype:wild-type | Sample source:CD8 T cell RAW_FILE_NAME(Raw file name)=WT1.RAW SUBJECT_SAMPLE_FACTORS S-1 WT2 Genotype:wild-type | Sample source:CD8 T cell RAW_FILE_NAME(Raw file name)=WT2.RAW SUBJECT_SAMPLE_FACTORS S-1 WT3 Genotype:wild-type | Sample source:CD8 T cell RAW_FILE_NAME(Raw file name)=WT3.RAW SUBJECT_SAMPLE_FACTORS S-1 WT4 Genotype:wild-type | Sample source:CD8 T cell RAW_FILE_NAME(Raw file name)=WT4.RAW SUBJECT_SAMPLE_FACTORS S-1 WT5 Genotype:wild-type | Sample source:CD8 T cell RAW_FILE_NAME(Raw file name)=WT5.RAW #COLLECTION CO:COLLECTION_SUMMARY Mouse CD8+ T cells were isolated from the spleen and lymph nodes of ME2 CO:COLLECTION_SUMMARY wild-type and ME2 knockout mice (knockdown of ME2 in T cells). The mice were CO:COLLECTION_SUMMARY euthanized and soaked in 75% alcohol for 3 minutes. Separate mouse spleens using CO:COLLECTION_SUMMARY a dissector and grind them into single-cell suspensions using a grinder. CO:COLLECTION_SUMMARY Single-cell suspension was used for CD8+ T cell isolation using the Mouse CO:COLLECTION_SUMMARY Lymphocyte Isolation Kit (Biolegend, 480044, China). T cells were cultured in CO:COLLECTION_SUMMARY RMPI-1640 medium supplemented with 10% FBS, 10μg/ml β-mercaptoethanol, and 1% CO:COLLECTION_SUMMARY penicillin and streptomycin. For metabolite analysis, CD8+ T cells were CO:COLLECTION_SUMMARY stimulated with 4 μg/ml anti-CD3 and 1μg/ml anti-CD28 antibodies for 24 h. CO:SAMPLE_TYPE T-cells CO:STORAGE_CONDITIONS On ice #TREATMENT TR:TREATMENT_SUMMARY Naive ME2+/+ and ME2-/- CD8+ T cells were isolated and activated with anti-CD3 TR:TREATMENT_SUMMARY (4μg/ml) and anti-CD28 (1μg/ml). #SAMPLEPREP SP:SAMPLEPREP_SUMMARY For metabolite analysis, naive ME2+/+ and ME2-/- CD8+ T cells were isolated and SP:SAMPLEPREP_SUMMARY activated with anti-CD3 (4μg/ml) and anti-CD28 (1μg/ml). Cells were washed SP:SAMPLEPREP_SUMMARY twice with ice-cold PBS and metabolites were extracted with ice-cold 80% SP:SAMPLEPREP_SUMMARY methanol. The extracts were analyzed by LC-MS/MS. SP:EXTRACTION_METHOD Cells were washed twice with ice-cold PBS and metabolites were extracted with SP:EXTRACTION_METHOD ice-cold 80% methanol. SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 CH:COLUMN_NAME Merck SeQuant ZIC-HILIC (150 x 2.1mm,5um) CH:COLUMN_TEMPERATURE 35 CH:FLOW_GRADIENT 0-2 min, 100% phase A; 2-11 min, 0% to 100% B; 11-13 min, 100% B; 13-15 min, 0% CH:FLOW_GRADIENT to 100% A. CH:FLOW_RATE 0.4 ml/min CH:SOLVENT_A 100% water; 0.1 % formic acid CH:SOLVENT_B 100% methanol; 0.1 % formic acid CH:CHROMATOGRAPHY_TYPE HILIC #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Focus MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:MS_COMMENTS The Q Exactive mass spectrometer was operated in full MS and polarity switching MS:MS_COMMENTS mode (acquisition from m/z 50 to 500) with a scan resolution set at 35,000. Five MS:MS_COMMENTS independent cell cultures were measured for each condition and samples were MS:MS_COMMENTS randomised in order to avoid bias in sample analyses due to machine drift. Q MS:MS_COMMENTS Exactive LC-MS/MS vendor raw data files were converted to the open source format MS:MS_COMMENTS mzML using the program ProteoWizard, and then the XCMS program was used for peak MS:MS_COMMENTS alignment, retention time correction and peak area extraction. Significance was MS:MS_COMMENTS determined using an unpaired Student's t-test. p-value < 0.05 was considered as MS:MS_COMMENTS statistically significant. The acquired spectra were analysed using XCalibur MS:MS_COMMENTS Qual Browser and XCalibur Quan Browser software (Thermo Scientific) by MS:MS_COMMENTS referencing to an internal library of compounds. MS:ION_MODE UNSPECIFIED #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Peak area MS_METABOLITE_DATA_START Samples K1 K2 K3 KO1 KO2 KO3 KO4 KO5 W1 W2 W3 WT1 WT2 WT3 WT4 WT5 Factors Genotype:Me2-knockout | Sample source:CD8 T cell Genotype:Me2-knockout | Sample source:CD8 T cell Genotype:Me2-knockout | Sample source:CD8 T cell Genotype:Me2-knockout | Sample source:CD8 T cell Genotype:Me2-knockout | Sample source:CD8 T cell Genotype:Me2-knockout | Sample source:CD8 T cell Genotype:Me2-knockout | Sample source:CD8 T cell Genotype:Me2-knockout | Sample source:CD8 T cell Genotype:wild-type | Sample source:CD8 T cell Genotype:wild-type | Sample source:CD8 T cell Genotype:wild-type | Sample source:CD8 T cell Genotype:wild-type | Sample source:CD8 T cell Genotype:wild-type | Sample source:CD8 T cell Genotype:wild-type | Sample source:CD8 T cell Genotype:wild-type | Sample source:CD8 T cell Genotype:wild-type | Sample source:CD8 T cell Adenosine Diphosphate(ADP) 5360302.0000 5281175.5000 4863602.5000 2170000.0000 3450000.0000 3490000.0000 2890000.0000 2730000.0000 3566545.2500 2635199.2500 3961358.7500 5370000.0000 3470000.0000 3200000.0000 3200000.0000 2560000.0000 Adenosine Monophosphate(AMP) 443554.9100 612365.5600 376734.5900 695000.0000 896000.0000 910000.0000 779000.0000 787000.0000 923732.8100 674310.5600 544652.6900 1150000.0000 914000.0000 825000.0000 720000.0000 722000.0000 Adenosine Triphosphate(ATP) 1016199.6900 1123779.7500 1393685.1200 23900000.0000 36000000.0000 31900000.0000 23200000.0000 20400000.0000 654364.0600 738914.5600 690311.4400 50900000.0000 30700000.0000 28400000.0000 29600000.0000 34300000.0000 Choline 112415192.0000 104843560.0000 122579224.0000 2060000.0000 1240000.0000 1770000.0000 1590000.0000 1970000.0000 109157376.0000 110414752.0000 108186024.0000 988000.0000 1310000.0000 1150000.0000 1600000.0000 2770000.0000 Citric Acid 831950.5000 1175230.3800 2267278.0000 1430000.0000 653000.0000 727000.0000 797000.0000 1750000.0000 400507.1600 170810.8000 1046639.5000 2010000.0000 840000.0000 768000.0000 2150000.0000 1160000.0000 Creatine 277272.1900 240844.3900 237221.8600 242000.0000 139000.0000 166000.0000 227000.0000 209000.0000 202860.9500 232698.5600 257040.9500 103000.0000 129000.0000 133000.0000 209000.0000 256000.0000 Fructose 1_6-Bisphosphate 85321.6600 72985.9900 53750.2200 173000.0000 192000.0000 157000.0000 132000.0000 198000.0000 48961.5000 57595.2500 47305.6900 443000.0000 229000.0000 170000.0000 284000.0000 191000.0000 Fumaric Acid 40910924.0000 39579252.0000 40370740.0000 650000.0000 811000.0000 736000.0000 753000.0000 712000.0000 3881326.0000 4596558.0000 4905779.0000 109000.0000 70400.0000 67700.0000 99700.0000 73300.0000 Gamma-Aminobutyric Acid 4787206.0000 4567318.0000 5074342.0000 357000.0000 194000.0000 298000.0000 261000.0000 335000.0000 5985165.5000 6824239.5000 6742473.5000 178000.0000 208000.0000 180000.0000 276000.0000 485000.0000 L-Lactic Acid 92217152.0000 65892508.0000 94951104.0000 384000.0000 165000.0000 108000.0000 223000.0000 308000.0000 71447112.0000 92646104.0000 69461024.0000 121000.0000 244000.0000 180000.0000 189000.0000 315000.0000 Malic Acid 334112480.0000 317340128.0000 317928064.0000 9810000.0000 11800000.0000 10700000.0000 10500000.0000 10300000.0000 30549206.0000 37206416.0000 41900972.0000 1830000.0000 810000.0000 817000.0000 1330000.0000 888000.0000 NAD 3626126.7500 4459451.5000 4102124.0000 1870000.0000 2030000.0000 1870000.0000 1830000.0000 2030000.0000 2667292.2500 2812191.2500 2899324.0000 2370000.0000 1550000.0000 1480000.0000 2090000.0000 1820000.0000 NADH 411093.0900 443840.3400 392185.6200 88000.0000 104000.0000 91500.0000 100000.0000 57800.0000 389966.5900 474420.0000 379897.9100 180000.0000 146000.0000 137000.0000 203000.0000 108000.0000 NADP 110334.0200 945000.0000 1320000.0000 1050000.0000 1010000.0000 871000.0000 3180.1600 94372.8100 1570000.0000 1060000.0000 862000.0000 1200000.0000 925000.0000 NADPH 53489.0300 112655.9400 33893.9000 36100.0000 25500.0000 42300.0000 4600.0000 5440.0000 40242.5200 49131.6200 11903.7400 83400.0000 5840.0000 101000.0000 7780.0000 55300.0000 Succinic Acid 12713237.0000 11103603.0000 13089128.0000 6030000.0000 5990000.0000 5640000.0000 6180000.0000 6890000.0000 6596342.5000 10191412.0000 7935360.0000 2900000.0000 1810000.0000 1730000.0000 2330000.0000 1910000.0000 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name pubchem_id inchi_key kegg_id other_id other_id_type ri ri_type moverz_quant Adenosine Diphosphate(ADP) C05543 Adenosine Monophosphate(AMP) C00186 Adenosine Triphosphate(ATP) C00300 Choline C00127 Citric Acid C00670 Creatine C00093 Fructose 1,6-Bisphosphate C00114 Fumaric Acid C01157 Gamma-Aminobutyric Acid C00021 L-Lactic Acid Malic Acid C00597 NAD C00119 NADH C00002 NADP C00148 NADPH C00112 Succinic Acid C00301 METABOLITES_END #END