{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002085","ANALYSIS_ID":"AN003403","VERSION":"1","CREATED_ON":"February 15, 2022, 5:44 am"},

"PROJECT":{"PROJECT_TITLE":"Proline metabolism in T cells","PROJECT_SUMMARY":"T cell metabolites detection after PRODH2 overexpression.","INSTITUTE":"Yale University","LAST_NAME":"Ye","FIRST_NAME":"Lupeng","ADDRESS":"850 West campus drive","EMAIL":"lupeng.ye@yale.edu","PHONE":"2035436568"},

"STUDY":{"STUDY_TITLE":"A genome-scale gain-of-function CRISPR screen in CD8 T cells identifies proline metabolism as a means to enhance CAR-T therapy(Part 1)","STUDY_SUMMARY":"Chimeric antigen receptor (CAR)-T cell-based immunotherapy for cancer and immunological diseases has made great strides, but it still faces multiple hurdles. Finding the right molecular targets to engineer T cells toward a desired function has broad implications for the armamentarium of T cell-centered therapies. Here, we developed a dead-guide RNA (dgRNA)-based CRISPR activation screen in primary CD8+ T cells, and identified gain-of-function (GOF) targets for CAR-T engineering. Targeted knock-in or overexpression of a lead target, PRODH2, enhanced CAR-T-based killing and in vivo efficacy in multiple cancer models. Transcriptomics and metabolomics in CAR-T cells revealed that augmenting PRODH2 expression re-shaped broad and distinct gene expression and metabolic programs. Mitochondrial, metabolic and immunological analyses showed that PRODH2 engineering enhances the metabolic and immune functions of CAR-T cells against cancer. Together these findings provide a system for identification of GOF immune boosters, and demonstrate PRODH2 as a target to enhance CAR-T efficacy.","INSTITUTE":"Yale University","LAST_NAME":"Ye","FIRST_NAME":"Lupeng","ADDRESS":"850 West campus drive","EMAIL":"lupeng.ye@yale.edu","PHONE":"2035436568"},

"SUBJECT":{"SUBJECT_TYPE":"Cultured cells","SUBJECT_SPECIES":"Homo sapiens","TAXONOMY_ID":"9606"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"20181221_hProdh2_OE_1_M62",
"Factors":{"Genotype":"PRODH2_OE","Treatment":"PRODH2"},
"Additional sample data":{"RAW_FILE_NAME":"20181221_hProdh2_OE_1_M62.d"}
},
{
"Subject ID":"-",
"Sample ID":"20181221_hProdh2_OE_2_M62",
"Factors":{"Genotype":"PRODH2_OE","Treatment":"PRODH2"},
"Additional sample data":{"RAW_FILE_NAME":"20181221_hProdh2_OE_2_M62.d"}
},
{
"Subject ID":"-",
"Sample ID":"20181221_hProdh2_OE_3_M62",
"Factors":{"Genotype":"PRODH2_OE","Treatment":"PRODH2"},
"Additional sample data":{"RAW_FILE_NAME":"20181221_hProdh2_OE_3_M62.d"}
},
{
"Subject ID":"-",
"Sample ID":"20181221_hProdh2_OE_4_M62",
"Factors":{"Genotype":"PRODH2_OE","Treatment":"PRODH2"},
"Additional sample data":{"RAW_FILE_NAME":"20181221_hProdh2_OE_4_M62.d"}
},
{
"Subject ID":"-",
"Sample ID":"20181221_hProdh2_OE_5_M62",
"Factors":{"Genotype":"PRODH2_OE","Treatment":"PRODH2"},
"Additional sample data":{"RAW_FILE_NAME":"20181221_hProdh2_OE_5_M62.d"}
},
{
"Subject ID":"-",
"Sample ID":"20181221_hProdh2_OE_Vector1_M62",
"Factors":{"Genotype":"Vector_OE","Treatment":"Control"},
"Additional sample data":{"RAW_FILE_NAME":"20181221_hProdh2_OE_Vector1_M62.d"}
},
{
"Subject ID":"-",
"Sample ID":"20181221_hProdh2_OE_Vector2_M62",
"Factors":{"Genotype":"Vector_OE","Treatment":"Control"},
"Additional sample data":{"RAW_FILE_NAME":"20181221_hProdh2_OE_Vector2_M62.d"}
},
{
"Subject ID":"-",
"Sample ID":"20181221_hProdh2_OE_Vector3_M62",
"Factors":{"Genotype":"Vector_OE","Treatment":"Control"},
"Additional sample data":{"RAW_FILE_NAME":"20181221_hProdh2_OE_Vector3_M62.d"}
},
{
"Subject ID":"-",
"Sample ID":"20181221_hProdh2_OE_Vector4_M62",
"Factors":{"Genotype":"Vector_OE","Treatment":"Control"},
"Additional sample data":{"RAW_FILE_NAME":"20181221_hProdh2_OE_Vector4_M62.d"}
},
{
"Subject ID":"-",
"Sample ID":"20181221_hProdh2_OE_Vector5_M62",
"Factors":{"Genotype":"Vector_OE","Treatment":"Control"},
"Additional sample data":{"RAW_FILE_NAME":"20181221_hProdh2_OE_Vector5_M62.d"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"cell were counted, washed twice with PBS then used for metabolite extraction.","SAMPLE_TYPE":"T-cells"},

"TREATMENT":{"TREATMENT_SUMMARY":"T cells were transduced with lentivirus, then collected for metabolite extraction."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"After normalizing cell counts, 800 μL of 80 % (vol / vol) HPLC-grade methanol (Sigma) (precooled to -80 oC on dry ice) was added to fresh cells in a 1.5-mL microcentrifuge tube, then tubes were put on dry ice for 30 minutes. The tubes were then incubated on ice for 20 minutes and centrifuged at 15,000 x g for 15 min at 4 oC to pellet the cell debris. The metabolite-containing supernatant was transferred to a new 1.5-mL microcentrifuge tube on dry ice. Metabolite extraction was repeated with 400 μL of 80 % (vol / vol) HPLC-grade methanol. The cell lysate / methanol mixtures were dried by Speedvac at room temperature. The metabolites were dissolved again with 80 % (vol / vol) methanol, then centrifuged at 18,000 x g for 10 min to remove any particulates, and the metabolite mixtures were stored at -80 oC until LC-MS analysis."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"HILIC","INSTRUMENT_NAME":"Agilent 6550","COLUMN_NAME":"Phenomenex Kinetex C18 (150 x 2.1mm, 2.6 um)"},

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

"MS":{"INSTRUMENT_NAME":"Agilent 6550 QTOF","INSTRUMENT_TYPE":"QTOF","MS_TYPE":"ESI","ION_MODE":"POSITIVE","MS_COMMENTS":"Two metabolomics strategies were adopted, i.e. untargeted metabolomics (aiming to unbiasedly detect all detectable metabolites) and targeted approaches (aiming to detect specifically defined metabolites, such as related metabolites in the proline metabolism and T cell metabolism). For untargeted metabolomics analysis, the optimized workflow consists of automated peak detection and integration, peak alignment, background noise subtraction, and multivariate data analysis. These steps were carried out for comprehensive metabolite phenotyping of the two groups using Agilent Mass Hunter Qualitative Analysis Software (Version B.07.0.0, build 7.0.7024.0) and Agilent Mass Profiler Professional (Version 14.5-Build 2772). The metabolites were first putatively identified based on accurate mass match (accurate mass ± 30 ppm error) and fragmentation pattern match. Putative structural annotation was carried out by searching the metabolite databases HMDB (http://www.hmdb.ca/) and METLIN (http:// metlin.scripps.edu) using the mass-to-charge ratio of the metabolic features."},

"MS_METABOLITE_DATA":{
"Units":"ppm",

"Data":[{"Metabolite":"Glutathione","20181221_hProdh2_OE_Vector1_M62":"921413","20181221_hProdh2_OE_Vector2_M62":"604123","20181221_hProdh2_OE_Vector3_M62":"487883","20181221_hProdh2_OE_Vector4_M62":"1620478","20181221_hProdh2_OE_Vector5_M62":"1019804","20181221_hProdh2_OE_1_M62":"1470537","20181221_hProdh2_OE_2_M62":"911867","20181221_hProdh2_OE_3_M62":"1597407","20181221_hProdh2_OE_4_M62":"762426","20181221_hProdh2_OE_5_M62":"1431081"},{"Metabolite":"Sphinganine","20181221_hProdh2_OE_Vector1_M62":"32621","20181221_hProdh2_OE_Vector2_M62":"25332","20181221_hProdh2_OE_Vector3_M62":"30326","20181221_hProdh2_OE_Vector4_M62":"39073","20181221_hProdh2_OE_Vector5_M62":"31656","20181221_hProdh2_OE_1_M62":"43878","20181221_hProdh2_OE_2_M62":"22062","20181221_hProdh2_OE_3_M62":"20403","20181221_hProdh2_OE_4_M62":"35196","20181221_hProdh2_OE_5_M62":"39059"},{"Metabolite":"Argininosuccinate","20181221_hProdh2_OE_Vector1_M62":"3149","20181221_hProdh2_OE_Vector2_M62":"1984","20181221_hProdh2_OE_Vector3_M62":"1603","20181221_hProdh2_OE_Vector4_M62":"3472","20181221_hProdh2_OE_Vector5_M62":"2733","20181221_hProdh2_OE_1_M62":"1235","20181221_hProdh2_OE_2_M62":"3082","20181221_hProdh2_OE_3_M62":"2542","20181221_hProdh2_OE_4_M62":"3363","20181221_hProdh2_OE_5_M62":"3183"},{"Metabolite":"Homocystine","20181221_hProdh2_OE_Vector1_M62":"473","20181221_hProdh2_OE_Vector2_M62":"449","20181221_hProdh2_OE_Vector3_M62":"875","20181221_hProdh2_OE_Vector4_M62":"1629","20181221_hProdh2_OE_Vector5_M62":"1120","20181221_hProdh2_OE_1_M62":"288","20181221_hProdh2_OE_2_M62":"2058","20181221_hProdh2_OE_3_M62":"1325","20181221_hProdh2_OE_4_M62":"984","20181221_hProdh2_OE_5_M62":"1394"},{"Metabolite":"Cystine","20181221_hProdh2_OE_Vector1_M62":"3314","20181221_hProdh2_OE_Vector2_M62":"490","20181221_hProdh2_OE_Vector3_M62":"1438","20181221_hProdh2_OE_Vector4_M62":"9123","20181221_hProdh2_OE_Vector5_M62":"3205","20181221_hProdh2_OE_1_M62":"6809","20181221_hProdh2_OE_2_M62":"7638","20181221_hProdh2_OE_3_M62":"10820","20181221_hProdh2_OE_4_M62":"10351","20181221_hProdh2_OE_5_M62":"5970"},{"Metabolite":"Anserine","20181221_hProdh2_OE_Vector1_M62":"6567","20181221_hProdh2_OE_Vector2_M62":"1036","20181221_hProdh2_OE_Vector3_M62":"2801","20181221_hProdh2_OE_Vector4_M62":"3031","20181221_hProdh2_OE_Vector5_M62":"1164","20181221_hProdh2_OE_1_M62":"1072","20181221_hProdh2_OE_2_M62":"2752","20181221_hProdh2_OE_3_M62":"1595","20181221_hProdh2_OE_4_M62":"2533","20181221_hProdh2_OE_5_M62":"792"},{"Metabolite":"Cystathionine","20181221_hProdh2_OE_Vector1_M62":"2440","20181221_hProdh2_OE_Vector2_M62":"1871","20181221_hProdh2_OE_Vector3_M62":"2619","20181221_hProdh2_OE_Vector4_M62":"2800","20181221_hProdh2_OE_Vector5_M62":"1226","20181221_hProdh2_OE_1_M62":"1167","20181221_hProdh2_OE_2_M62":"1907","20181221_hProdh2_OE_3_M62":"2336","20181221_hProdh2_OE_4_M62":"3702","20181221_hProdh2_OE_5_M62":"1116"},{"Metabolite":"Tryptophan","20181221_hProdh2_OE_Vector1_M62":"14206","20181221_hProdh2_OE_Vector2_M62":"17648","20181221_hProdh2_OE_Vector3_M62":"19085","20181221_hProdh2_OE_Vector4_M62":"16338","20181221_hProdh2_OE_Vector5_M62":"11956","20181221_hProdh2_OE_1_M62":"19105","20181221_hProdh2_OE_2_M62":"16018","20181221_hProdh2_OE_3_M62":"17618","20181221_hProdh2_OE_4_M62":"16552","20181221_hProdh2_OE_5_M62":"26498"},{"Metabolite":"Spermine","20181221_hProdh2_OE_Vector1_M62":"42471","20181221_hProdh2_OE_Vector2_M62":"36872","20181221_hProdh2_OE_Vector3_M62":"159882","20181221_hProdh2_OE_Vector4_M62":"221133","20181221_hProdh2_OE_Vector5_M62":"87789","20181221_hProdh2_OE_1_M62":"265372","20181221_hProdh2_OE_2_M62":"139276","20181221_hProdh2_OE_3_M62":"76612","20181221_hProdh2_OE_4_M62":"48855","20181221_hProdh2_OE_5_M62":"43190"},{"Metabolite":"3-Phosphoserine","20181221_hProdh2_OE_Vector1_M62":"2051","20181221_hProdh2_OE_Vector2_M62":"722","20181221_hProdh2_OE_Vector3_M62":"2442","20181221_hProdh2_OE_Vector4_M62":"2808","20181221_hProdh2_OE_Vector5_M62":"646","20181221_hProdh2_OE_1_M62":"1968","20181221_hProdh2_OE_2_M62":"3001","20181221_hProdh2_OE_3_M62":"878","20181221_hProdh2_OE_4_M62":"6420","20181221_hProdh2_OE_5_M62":"2976"},{"Metabolite":"Tyrosne","20181221_hProdh2_OE_Vector1_M62":"264182","20181221_hProdh2_OE_Vector2_M62":"42504","20181221_hProdh2_OE_Vector3_M62":"61903","20181221_hProdh2_OE_Vector4_M62":"73028","20181221_hProdh2_OE_Vector5_M62":"43442","20181221_hProdh2_OE_1_M62":"75233","20181221_hProdh2_OE_2_M62":"44268","20181221_hProdh2_OE_3_M62":"47975","20181221_hProdh2_OE_4_M62":"46778","20181221_hProdh2_OE_5_M62":"49910"},{"Metabolite":"Citrulline","20181221_hProdh2_OE_Vector1_M62":"1853","20181221_hProdh2_OE_Vector2_M62":"2570","20181221_hProdh2_OE_Vector3_M62":"1890","20181221_hProdh2_OE_Vector4_M62":"2845","20181221_hProdh2_OE_Vector5_M62":"1435","20181221_hProdh2_OE_1_M62":"2259","20181221_hProdh2_OE_2_M62":"9008","20181221_hProdh2_OE_3_M62":"7373","20181221_hProdh2_OE_4_M62":"2752","20181221_hProdh2_OE_5_M62":"3765"},{"Metabolite":"Arginine","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OE_4_M62":"93700","20181221_hProdh2_OE_5_M62":"273430"},{"Metabolite":"Malate","20181221_hProdh2_OE_Vector1_M62":"3476","20181221_hProdh2_OE_Vector2_M62":"5557","20181221_hProdh2_OE_Vector3_M62":"5757","20181221_hProdh2_OE_Vector4_M62":"5807","20181221_hProdh2_OE_Vector5_M62":"5391","20181221_hProdh2_OE_1_M62":"2466","20181221_hProdh2_OE_2_M62":"1588","20181221_hProdh2_OE_3_M62":"3451","20181221_hProdh2_OE_4_M62":"4448","20181221_hProdh2_OE_5_M62":"2989"},{"Metabolite":"Aspartic 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}

}