#METABOLOMICS WORKBENCH parthosen_20210131_174239 DATATRACK_ID:2440 STUDY_ID:ST001679 ANALYSIS_ID:AN002737 PROJECT_ID:000000 VERSION 1 CREATED_ON February 3, 2021, 9:31 am #PROJECT PR:PROJECT_TITLE Quantitative analysis and genome-scale modeling of human CD4+ T-cell PR:PROJECT_TITLE differentiation reveals subset-specific regulation of glycosphingolipid pathways PR:PROJECT_TYPE MS: Untargeted and Targeted PR:PROJECT_SUMMARY This project is associated with five different studies(Part 1-5) and each study PR:PROJECT_SUMMARY is associated with one dataset. All the datasets are submitted to Metabolomics PR:PROJECT_SUMMARY Workbench. Part 1/5: It includes untargeted lipidomic analysis of CD4+ T-cell PR:PROJECT_SUMMARY subsets(Th1,Th2,Th17 and iTreg cells) and their paired control(Th0)cells. Part PR:PROJECT_SUMMARY 2/5: It includes quantitative targeted measurements of sphingolipids(ceramides PR:PROJECT_SUMMARY and glycosphingolipids) in Th17, iTreg, and their paired control (Th0) cells. PR:PROJECT_SUMMARY Part 3/5: It includes quantitative targeted measurements of PR:PROJECT_SUMMARY sphingolipids(ceramides and glycosphingolipids) in Th17 cells before(scrambled / PR:PROJECT_SUMMARY control) and after the triple knockdown of SPTLC1,2,3 genes (SPT de novo PR:PROJECT_SUMMARY pathway: sphingolipid metabolism). Part 4/5: It includes quantitative targeted PR:PROJECT_SUMMARY measurements of sphingolipids(ceramides, glycosphingolipids) in Th17 cells PR:PROJECT_SUMMARY before (scrambled / control)and after the knockdown of UGCG gene (GCS pathway: PR:PROJECT_SUMMARY sphingolipid metabolism). Part 5/5: It includes measurements of sphingolipids PR:PROJECT_SUMMARY (sphingomyelins) in Th17 cells before (scrambled / control)and after the PR:PROJECT_SUMMARY knockdown of UGCG gene(GCS pathway: sphingolipid metabolism). PR:INSTITUTE University of Turku PR:DEPARTMENT Systems Medicine, Turku Bioscience PR:LABORATORY Metabolomics PR:LAST_NAME Sen PR:FIRST_NAME Partho PR:ADDRESS Tykistökatu 6B, BioCity, 5th Floor, Turku, Southwest, 20521, Finland PR:EMAIL partho.sen@utu.fi PR:PHONE 0469608145 #STUDY ST:STUDY_TITLE Quantitative measurements of sphingomyelins in Th17 cells before and after the ST:STUDY_TITLE knockdown of UGCG gene (GCS pathway: sphingolipid metabolism) (part-V) ST:STUDY_TYPE MS: Untargeted and targeted analysis ST:STUDY_SUMMARY Part 5/5: It includes measurements of sphingolipids (sphingomyelins) in Th17 ST:STUDY_SUMMARY cells before (scrambled / control) and after the knockdown of UGCG gene (GCS ST:STUDY_SUMMARY pathway: sphingolipid metabolism). ST:INSTITUTE University of Turku ST:DEPARTMENT Systems Medicine, Turku Bioscience ST:LABORATORY Metabolomics ST:LAST_NAME Sen ST:FIRST_NAME Partho ST:ADDRESS Tykistökatu 6B, BioCity, 5th Floor, Turku, Southwest, 20521, Finland ST:EMAIL partho.sen@utu.fi ST:PHONE 0469608145 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:GENDER Not applicable #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - SCr-D1 Treatment:Scrambled | Types:Control Cell Type=T17; RAW_FILE_NAME=SCr-D1.mzML SUBJECT_SAMPLE_FACTORS - KD-D1 Treatment:UGCG-knockdown | Types:Test Cell Type=T17; RAW_FILE_NAME=KD-D1.mzML SUBJECT_SAMPLE_FACTORS - SCr-D2 Treatment:Scrambled | Types:Control Cell Type=T17; RAW_FILE_NAME=SCr-D2.mzML SUBJECT_SAMPLE_FACTORS - KD-D2 Treatment:UGCG-knockdown | Types:Test Cell Type=T17; RAW_FILE_NAME=KD-D2.mzML SUBJECT_SAMPLE_FACTORS - SCr-D3 Treatment:Scrambled | Types:Control Cell Type=T17; RAW_FILE_NAME=SCr-D3.mzML SUBJECT_SAMPLE_FACTORS - KD-D3 Treatment:UGCG-knockdown | Types:Test Cell Type=T17; RAW_FILE_NAME=KD-D3.mzML #COLLECTION CO:COLLECTION_SUMMARY CD4+ T-cells were isolated from human umbilical cord blood as described CO:COLLECTION_SUMMARY previously[1-3]. 1. Ubaid, U. et al. Transcriptional Repressor HIC1 Contributes CO:COLLECTION_SUMMARY to Suppressive Function of Human Induced Regulatory T Cells. Cell Rep 22, CO:COLLECTION_SUMMARY 2094-2106, doi:10.1016/j.celrep.2018.01.070 (2018). 2. Khan, M. M. et al. CIP2A CO:COLLECTION_SUMMARY Constrains Th17 Differentiation by Modulating STAT3 Signaling. iScience 23, CO:COLLECTION_SUMMARY 100947, doi:10.1016/j.isci.2020.100947 (2020). 3. Tripathi, S. K. et al. CO:COLLECTION_SUMMARY Genome-wide Analysis of STAT3-Mediated Transcription during Early Human Th17 CO:COLLECTION_SUMMARY Cell Differentiation. Cell Rep 19, 1888-1901, doi:10.1016/j.celrep.2017.05.013 CO:COLLECTION_SUMMARY (2017). CO:SAMPLE_TYPE T-cells CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY For Th17 cell differentiation, isolated CD4+ cells were activated with a TR:TREATMENT_SUMMARY combination of plate-bound anti-CD3 (750 ng/24-well culture plate well; TR:TREATMENT_SUMMARY Immunotech/Beckman Coulter REF # IM-1304) and soluble anti-CD28 ((1ug/mL; TR:TREATMENT_SUMMARY Immunotech/Beckman coulter REF # IM1376) antibodies in serum-free X-Vivo 20 TR:TREATMENT_SUMMARY medium (Lonza), in the absence (Th0) or presence (Th17) of IL-6 (20ng/ml, Roche, TR:TREATMENT_SUMMARY Cat# 11138600 001); IL-1β (10ng/ml, R&D Systems Cat # 201 LB); TGF-β1 TR:TREATMENT_SUMMARY (10ng/ml, R&D Systems Cat# 240); anti-IL-4 (1 g/ml) R&D Systems Cat# MAB204) TR:TREATMENT_SUMMARY and anti-IFN-γ (1 μg/ml R&D Systems Cat#MAB-285). Differentiation of Th17 TR:TREATMENT_SUMMARY cells was confirmed by measuring IL-17 expression by quantitative real-time PCR, TR:TREATMENT_SUMMARY at 72 hours of Th17 / Th0 culturing. For iTreg cell culturing, after of CD25+ TR:TREATMENT_SUMMARY cells, done using LD columns and a CD25 depletion kit (Miltenyi Biotec), TR:TREATMENT_SUMMARY CD4+CD25− cells were activated with plate-bound anti-CD3 (500 ng/24-well TR:TREATMENT_SUMMARY culture plate well) and soluble anti-CD28 (500 ng/mL) at a density of 2 × 106 TR:TREATMENT_SUMMARY cells/mL of X-vivo 15 serum-free medium (Lonza). For iTreg differentiation, the TR:TREATMENT_SUMMARY medium was supplemented with IL-2 (12 ng/mL), TGF-β (10 ng/mL) (both from R&D TR:TREATMENT_SUMMARY Systems), all-trans retinoic acid (ATRA) (10 nM; Sigma-Aldrich), and human serum TR:TREATMENT_SUMMARY (10%) and cultured at 37°C in 5% CO2. Control Th0 cells were stimulated with TR:TREATMENT_SUMMARY plate-bound anti-CD3 soluble anti-CD28 antibodies without cytokines. For TR:TREATMENT_SUMMARY confirmation of iTreg cell differentiation, we used intracellular staining to TR:TREATMENT_SUMMARY measure, at 72 hours of iTreg culturing, expression of FOXP3 which is the major TR:TREATMENT_SUMMARY transcription factor driving Treg differentiation. Intracellular staining was TR:TREATMENT_SUMMARY performed using buffer sets of Human Regulatory T-cell Staining Kit TR:TREATMENT_SUMMARY (eBioscience/Thermo Fisher Scientific), following the manufacturer’s protocol. TR:TREATMENT_SUMMARY The following antibodies were used: anti-human FOXP3-PE (eBioscience, Cat. No. TR:TREATMENT_SUMMARY 12-4776-42) and rat IgG2a isotype control (eBioscience, Cat. No. 72-4321-77A). TR:TREATMENT_SUMMARY All samples were acquired by a flow cytometer (LSRII) and analyzed either with TR:TREATMENT_SUMMARY FlowJo (FLOWJO, LLC) or with Flowing Software. For Th1 and Th2 cells, purified TR:TREATMENT_SUMMARY naive CD4+ T-cells were activated with plate-bound anti-CD3 (500 ng/24-well TR:TREATMENT_SUMMARY culture plate well) and 500 ng/ml soluble anti-CD28 and cultured in the absence TR:TREATMENT_SUMMARY (Th0) or presence of 2.5 ng/ml IL-12 (R&D Systems) (Th1) or 10 ng/ml IL-4 (R&D TR:TREATMENT_SUMMARY Systems) (for Th2). At 48 hours following the activation of the cells, 17 ng/ml TR:TREATMENT_SUMMARY IL-2 (R&D Systems) was added to the cultures. Differentiation of Th1 and Th2 TR:TREATMENT_SUMMARY cells was confirmed by measuring (using flow cytometry) the expression of T-bet TR:TREATMENT_SUMMARY and Gata3 at 72 hours after cell activation. Briefly, cells were fixed and TR:TREATMENT_SUMMARY permeabilized using the Intracellular Fixation & Permeabilization Buffer Set TR:TREATMENT_SUMMARY (eBioscience / Thermo Fisher Scientific), according the manufacturer’s TR:TREATMENT_SUMMARY protocol. The following antibodies were used: anti-human GATA3-PE (eBioscience, TR:TREATMENT_SUMMARY 12-9966), anti-human T-bet-BV711 (BD, 563320) and corresponding isotype controls TR:TREATMENT_SUMMARY (BV711 Mouse IgG1, BD, 563044 and PE Rat IgG2b, eBioscience, 12-4031-82). TR:TREATMENT_SUMMARY Samples were acquired by BD LSRFortessa™ cell analyzer and data were analyzed TR:TREATMENT_SUMMARY using FlowJo software (FLOWJO, LLC). #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The frozen cell preps were defrosted on ice. The samples were extracted using a SP:SAMPLEPREP_SUMMARY modified Folch method[1]. 1. Sen, P. et al. Persistent Alterations in Plasma SP:SAMPLEPREP_SUMMARY Lipid Profiles Before Introduction of Gluten in the Diet Associated With SP:SAMPLEPREP_SUMMARY Progression to Celiac Disease. Clin Transl Gastroenterol 10, 1-10, SP:SAMPLEPREP_SUMMARY doi:10.14309/ctg.0000000000000044 (2019). Briefly, 120 µL of cold (4 °C) SP:SAMPLEPREP_SUMMARY extraction solvent (CHCl3: MeOH, (2:1 v/v) was added to the samples. The SP:SAMPLEPREP_SUMMARY extraction solvent containing the following internal standards: C17 SP:SAMPLEPREP_SUMMARY Lactosyl(beta) ceramide (D18:1/17:0, 20 ppb), C17 Glucosyl(beta) ceramide SP:SAMPLEPREP_SUMMARY (D18:1/17:0, 20 ppb), C17 ceramide (D18:1/17:0, 20 ppb), C16 ceramide-d7 SP:SAMPLEPREP_SUMMARY (d18:1-d7/16:0, 16,57 ppb), C18 ceramide-d7 (d18:1-d7/18:0, 8.75 ppb), C24 SP:SAMPLEPREP_SUMMARY ceramide-d7 (d18:1-d7/24:0, 20 ppb), and C24:1 ceramide-d7 (d18:1-d7/24:1(15Z), SP:SAMPLEPREP_SUMMARY 9,96 ppb). The samples were the vortexed briefly and left on ice for 30 minutes. SP:SAMPLEPREP_SUMMARY The samples were then centrifuged (9400g, 5 min, 4 °C) and then 60 µL of the SP:SAMPLEPREP_SUMMARY bottom layer was transfer to a clean mass spectrometry vial (2 mL). The samples SP:SAMPLEPREP_SUMMARY were then stored at –80 °C. The samples for this experiments were the same SP:SAMPLEPREP_SUMMARY extracts that the Cer data was acquired from and had SM(18:1/17:0) spiked in SP:SAMPLEPREP_SUMMARY prior to acquisition. SP:PROCESSING_STORAGE_CONDITIONS -80℃ SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Chromatographic separation was performed on an ACQUITY UHPLC BEH C18 column (2.1 CH:CHROMATOGRAPHY_SUMMARY mm × 100 mm, particle size 1.7 µm, Waters, Milford, MA, USA). The flow rate CH:CHROMATOGRAPHY_SUMMARY was set at 0.4 ml/min throughout the run with an injection volume of 1 µL. The CH:CHROMATOGRAPHY_SUMMARY following solvents were used for the gradient elution: Solvent A was H2O with 1% CH:CHROMATOGRAPHY_SUMMARY NH4Ac (1M) and HCOOH (0.1%) added. Solvent B was a mixture of ACN: IPA (1:1 v/v) CH:CHROMATOGRAPHY_SUMMARY with 1% NH4Ac (1M) and HCOOH (0.1%) added. The gradient was programmed as CH:CHROMATOGRAPHY_SUMMARY follows: 0 to 2 min 35-80% B, 2 to 7 min 80-100 % B, 7 to 14 min 100% B. The CH:CHROMATOGRAPHY_SUMMARY column was equilibrated with a 7min period of 35 % B prior to the next run. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters Acquity UHPLC UNSPSC 41115709 CH:COLUMN_NAME Waters BEH C18 (2.1 mm × 100 mm, 1.7 µm ) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Bruker impact II UHPLC-QTOF system MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS The SM results for UGCG-silenced Th17 cells data was acquired on a UHPLC-QTOF MS:MS_COMMENTS system from Bruker (Bruker, Billerica, MA, USA) combining an Elute UHPLC binary MS:MS_COMMENTS pump and an Impact II system QTOF system. The samples for this experiments were MS:MS_COMMENTS the same extracts that the Cer data was acquired from and had SM(18:1/17:0) MS:MS_COMMENTS spiked in prior to acquisition. The data was acquired using the Hystar suite of MS:MS_COMMENTS software. MZmine 2 was used for all the untargeted data processing. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS ng/ml MS_METABOLITE_DATA_START Samples SCr-D1 KD-D1 SCr-D2 KD-D2 SCr-D3 KD-D3 Factors Treatment:Scrambled | Types:Control Treatment:UGCG-knockdown | Types:Test Treatment:Scrambled | Types:Control Treatment:UGCG-knockdown | Types:Test Treatment:Scrambled | Types:Control Treatment:UGCG-knockdown | Types:Test SM(40:2) 17.20193205 29.92518117 17.13790764 30.84444737 31.18175593 16.6790407 SM(d39:1) 15.38197985 21.49159303 10.31057802 19.69177511 5.310709225 24.27979387 SM(d42:3) 45.39569106 80.55755523 47.76495437 89.8564094 106.0340446 38.43533259 SM(d34:2) 13.42104561 20.56499258 13.92247586 21.65716417 20.5471264 10.03529777 SM(d18:0/14:0) 4.349973139 6.253309045 4.631394023 7.272072629 6.791737851 3.835228151 SM(d18:1/16:0) 1304.095013 2122.820665 1269.762231 2318.336646 2209.709313 1537.240129 SM(d18:1/24:0) 141.8925289 216.5551407 122.029665 260.1155001 158.6334128 182.1644583 SM(d18:2/24:1) 1.544120503 2.486120427 1.59401539 3.413540223 2.999495106 1.042457274 SM(d32:1) 46.45775613 67.64853456 46.50944152 70.63184664 79.71227573 45.28629093 SM(d33:1) 12.41136815 16.410371 9.863641453 19.59395256 12.384952 17.05606495 SM(d36:1) 127.1885507 187.3470347 111.4172698 216.4558915 146.6283841 144.5802687 SM(d36:2) 7.312437134 11.58058873 6.561771229 8.85432372 7.673800181 7.391628972 SM(d38:2) 2.219475046 3.276988432 2.708171565 3.966326677 5.706922752 2.080261274 SM(d40:1) 147.964426 237.8186666 147.5238876 288.3692518 163.594511 219.4377597 SM(d41:1) 35.13400258 40.66125672 25.71612436 36.95160133 18.61520159 49.42096797 SM(d42:1) 11.55854829 20.9532022 13.78408087 24.36419072 18.10161024 10.42244865 SM(d42:2) 174.692472 311.313033 193.3236548 370.87741 391.5863064 168.6153408 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name quantified m/z RT(minutes) PubChem ID KEGG ID SM(40:2) 785.6485519 6.743978333 SM(d39:1) 773.6486401 7.003965 SM(d42:3) 811.6639938 6.739763333 SM(d34:2) 701.5547943 5.495344167 SM(d18:0/14:0) 677.5545912 5.536829167 SM(d18:1/16:0) 705.5847473 6.077355833 SM(d18:1/24:0) 815.6952006 7.538965833 SM(d18:2/24:1) 811.6638714 6.525891667 SM(d32:1) 675.5390422 5.327025833 SM(d33:1) 689.5547545 5.609240833 SM(d36:1) 731.6017965 6.380199167 SM(d36:2) 729.5850214 5.895625 SM(d38:2) 757.6174533 6.373169167 SM(d40:1) 787.6641947 7.1947175 SM(d41:1) 801.6798416 7.372094167 SM(d42:1) 815.694201 7.2714375 SM(d42:2) 813.6797969 7.122323333 METABOLITES_END #END