Summary of Study ST001679

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001078. The data can be accessed directly via it's Project DOI: 10.21228/M8C111 This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

Study ID	ST001679
Study Title	Quantitative measurements of sphingomyelins in Th17 cells before and after the knockdown of UGCG gene (GCS pathway: sphingolipid metabolism) (part-V)
Study Type	MS: Untargeted and targeted analysis
Study Summary	Part 5/5: It includes measurements of sphingolipids (sphingomyelins) in Th17 cells before (scrambled / control) and after the knockdown of UGCG gene (GCS pathway: sphingolipid metabolism).
Institute	University of Turku
Department	Systems Medicine, Turku Bioscience
Laboratory	Metabolomics
Last Name	Sen
First Name	Partho
Address	Tykistökatu 6B, BioCity, 5th Floor, Turku, Southwest, 20521, Finland
Email	partho.sen@utu.fi
Phone	0469608145
Submit Date	2021-01-31
Raw Data Available	Yes
Raw Data File Type(s)	mzML
Analysis Type Detail	LC-MS
Release Date	2021-11-02
Release Version	1

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Project:

Project ID:	PR001078
Project DOI:	doi: 10.21228/M8C111
Project Title:	Quantitative analysis and genome-scale modeling of human CD4+ T-cell differentiation reveals subset-specific regulation of glycosphingolipid pathways
Project Type:	MS: Targeted analysis
Project Summary:	This project is associated with five different studies(Part 1-5) and each study is associated with one dataset. All the datasets are submitted to Metabolomics Workbench. Part 1/5: It includes untargeted lipidomic analysis of CD4+ T-cell subsets (Th1,Th2,Th17 and iTreg cells) and their paired control (Th0) cells. Part 2/5: It includes quantitative targeted measurements of sphingolipids (ceramides and glycosphingolipids) in Th17, iTreg, and their paired control (Th0) cells. Part 3/5: It includes quantitative targeted measurements of sphingolipids (ceramides and glycosphingolipids) in Th17 cells before (scrambled / control) and after the triple knockdown of SPTLC1,2,3 genes (SPT de novo pathway: sphingolipid metabolism). Part 4/5: It includes quantitative targeted measurements of sphingolipids (ceramides, glycosphingolipids) in Th17 cells before (scrambled / control) and after the knockdown of UGCG gene (GCS pathway: sphingolipid metabolism). Part 5/5: It includes measurements of sphingolipids (sphingomyelins) in Th17 cells before (scrambled / control) and after the knockdown of UGCG gene(GCS pathway: sphingolipid metabolism).
Institute:	University of Turku
Department:	Systems Medicine
Laboratory:	Metabolomics
Last Name:	Sen
First Name:	Partho
Address:	Tykistökatu 6B, BioCity, 5th Floor, Turku, Southwest, 20521, Finland
Email:	partho.sen@utu.fi
Phone:	0469608145

Subject:

Subject ID:	SU001756
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	Not applicable

Factors:

Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id	local_sample_id	Treatment	Types
SA154561	SCr-D3	Scrambled	Control
SA154562	SCr-D1	Scrambled	Control
SA154563	SCr-D2	Scrambled	Control
SA154564	KD-D3	UGCG-knockdown	Test
SA154565	KD-D2	UGCG-knockdown	Test
SA154566	KD-D1	UGCG-knockdown	Test

Showing results 1 to 6 of 6

Showing results 1 to 6 of 6

Collection:

Collection ID:	CO001749
Collection Summary:	CD4+ T-cells were isolated from human umbilical cord blood as described previously[1-3]. 1. Ubaid, U. et al. Transcriptional Repressor HIC1 Contributes to Suppressive Function of Human Induced Regulatory T Cells. Cell Rep 22, 2094-2106, doi:10.1016/j.celrep.2018.01.070 (2018). 2. Khan, M. M. et al. CIP2A Constrains Th17 Differentiation by Modulating STAT3 Signaling. iScience 23, 100947, doi:10.1016/j.isci.2020.100947 (2020). 3. Tripathi, S. K. et al. Genome-wide Analysis of STAT3-Mediated Transcription during Early Human Th17 Cell Differentiation. Cell Rep 19, 1888-1901, doi:10.1016/j.celrep.2017.05.013 (2017).
Sample Type:	T-cells
Storage Conditions:	-80℃

Treatment:

Treatment ID:	TR001769
Treatment Summary:	For Th17 cell differentiation, isolated CD4+ cells were activated with a combination of plate-bound anti-CD3 (750 ng/24-well culture plate well; Immunotech/Beckman Coulter REF # IM-1304) and soluble anti-CD28 ((1ug/mL; Immunotech/Beckman coulter REF # IM1376) antibodies in serum-free X-Vivo 20 medium (Lonza), in the absence (Th0) or presence (Th17) of IL-6 (20ng/ml, Roche, Cat# 11138600 001); IL-1β (10ng/ml, R&D Systems Cat # 201 LB); TGF-β1 (10ng/ml, R&D Systems Cat# 240); anti-IL-4 (1 g/ml) R&D Systems Cat# MAB204) and anti-IFN-γ (1 μg/ml R&D Systems Cat#MAB-285). Differentiation of Th17 cells was confirmed by measuring IL-17 expression by quantitative real-time PCR, at 72 hours of Th17 / Th0 culturing. For iTreg cell culturing, after of CD25+ cells, done using LD columns and a CD25 depletion kit (Miltenyi Biotec), CD4+CD25− cells were activated with plate-bound anti-CD3 (500 ng/24-well culture plate well) and soluble anti-CD28 (500 ng/mL) at a density of 2 × 106 cells/mL of X-vivo 15 serum-free medium (Lonza). For iTreg differentiation, the medium was supplemented with IL-2 (12 ng/mL), TGF-β (10 ng/mL) (both from R&D Systems), all-trans retinoic acid (ATRA) (10 nM; Sigma-Aldrich), and human serum (10%) and cultured at 37°C in 5% CO2. Control Th0 cells were stimulated with plate-bound anti-CD3 soluble anti-CD28 antibodies without cytokines. For confirmation of iTreg cell differentiation, we used intracellular staining to measure, at 72 hours of iTreg culturing, expression of FOXP3 which is the major transcription factor driving Treg differentiation. Intracellular staining was performed using buffer sets of Human Regulatory T-cell Staining Kit (eBioscience/Thermo Fisher Scientific), following the manufacturer’s protocol. The following antibodies were used: anti-human FOXP3-PE (eBioscience, Cat. No. 12-4776-42) and rat IgG2a isotype control (eBioscience, Cat. No. 72-4321-77A). All samples were acquired by a flow cytometer (LSRII) and analyzed either with FlowJo (FLOWJO, LLC) or with Flowing Software. For Th1 and Th2 cells, purified naive CD4+ T-cells were activated with plate-bound anti-CD3 (500 ng/24-well culture plate well) and 500 ng/ml soluble anti-CD28 and cultured in the absence (Th0) or presence of 2.5 ng/ml IL-12 (R&D Systems) (Th1) or 10 ng/ml IL-4 (R&D Systems) (for Th2). At 48 hours following the activation of the cells, 17 ng/ml IL-2 (R&D Systems) was added to the cultures. Differentiation of Th1 and Th2 cells was confirmed by measuring (using flow cytometry) the expression of T-bet and Gata3 at 72 hours after cell activation. Briefly, cells were fixed and permeabilized using the Intracellular Fixation & Permeabilization Buffer Set (eBioscience / Thermo Fisher Scientific), according the manufacturer’s protocol. The following antibodies were used: anti-human GATA3-PE (eBioscience, 12-9966), anti-human T-bet-BV711 (BD, 563320) and corresponding isotype controls (BV711 Mouse IgG1, BD, 563044 and PE Rat IgG2b, eBioscience, 12-4031-82). Samples were acquired by BD LSRFortessa™ cell analyzer and data were analyzed using FlowJo software (FLOWJO, LLC).

Treatment ID:

TR001769

Treatment Summary:

For Th17 cell differentiation, isolated CD4+ cells were activated with a combination of plate-bound anti-CD3 (750 ng/24-well culture plate well; Immunotech/Beckman Coulter REF # IM-1304) and soluble anti-CD28 ((1ug/mL; Immunotech/Beckman coulter REF # IM1376) antibodies in serum-free X-Vivo 20 medium (Lonza), in the absence (Th0) or presence (Th17) of IL-6 (20ng/ml, Roche, Cat# 11138600 001); IL-1β (10ng/ml, R&D Systems Cat # 201 LB); TGF-β1 (10ng/ml, R&D Systems Cat# 240); anti-IL-4 (1 g/ml) R&D Systems Cat# MAB204) and anti-IFN-γ (1 μg/ml R&D Systems Cat#MAB-285). Differentiation of Th17 cells was confirmed by measuring IL-17 expression by quantitative real-time PCR, at 72 hours of Th17 / Th0 culturing. For iTreg cell culturing, after of CD25+ cells, done using LD columns and a CD25 depletion kit (Miltenyi Biotec), CD4+CD25− cells were activated with plate-bound anti-CD3 (500 ng/24-well culture plate well) and soluble anti-CD28 (500 ng/mL) at a density of 2 × 106 cells/mL of X-vivo 15 serum-free medium (Lonza). For iTreg differentiation, the medium was supplemented with IL-2 (12 ng/mL), TGF-β (10 ng/mL) (both from R&D Systems), all-trans retinoic acid (ATRA) (10 nM; Sigma-Aldrich), and human serum (10%) and cultured at 37°C in 5% CO2. Control Th0 cells were stimulated with plate-bound anti-CD3 soluble anti-CD28 antibodies without cytokines. For confirmation of iTreg cell differentiation, we used intracellular staining to measure, at 72 hours of iTreg culturing, expression of FOXP3 which is the major transcription factor driving Treg differentiation. Intracellular staining was performed using buffer sets of Human Regulatory T-cell Staining Kit (eBioscience/Thermo Fisher Scientific), following the manufacturer’s protocol. The following antibodies were used: anti-human FOXP3-PE (eBioscience, Cat. No. 12-4776-42) and rat IgG2a isotype control (eBioscience, Cat. No. 72-4321-77A). All samples were acquired by a flow cytometer (LSRII) and analyzed either with FlowJo (FLOWJO, LLC) or with Flowing Software. For Th1 and Th2 cells, purified naive CD4+ T-cells were activated with plate-bound anti-CD3 (500 ng/24-well culture plate well) and 500 ng/ml soluble anti-CD28 and cultured in the absence (Th0) or presence of 2.5 ng/ml IL-12 (R&D Systems) (Th1) or 10 ng/ml IL-4 (R&D Systems) (for Th2). At 48 hours following the activation of the cells, 17 ng/ml IL-2 (R&D Systems) was added to the cultures. Differentiation of Th1 and Th2 cells was confirmed by measuring (using flow cytometry) the expression of T-bet and Gata3 at 72 hours after cell activation. Briefly, cells were fixed and permeabilized using the Intracellular Fixation & Permeabilization Buffer Set (eBioscience / Thermo Fisher Scientific), according the manufacturer’s protocol. The following antibodies were used: anti-human GATA3-PE (eBioscience, 12-9966), anti-human T-bet-BV711 (BD, 563320) and corresponding isotype controls (BV711 Mouse IgG1, BD, 563044 and PE Rat IgG2b, eBioscience, 12-4031-82). Samples were acquired by BD LSRFortessa™ cell analyzer and data were analyzed using FlowJo software (FLOWJO, LLC).

Sample Preparation:

Sampleprep ID:	SP001762
Sampleprep Summary:	The frozen cell preps were defrosted on ice. The samples were extracted using a modified Folch method[1]. 1. Sen, P. et al. Persistent Alterations in Plasma Lipid Profiles Before Introduction of Gluten in the Diet Associated With Progression to Celiac Disease. Clin Transl Gastroenterol 10, 1-10, doi:10.14309/ctg.0000000000000044 (2019). Briefly, 120 µL of cold (4 °C) extraction solvent (CHCl3: MeOH, (2:1 v/v) was added to the samples. The extraction solvent containing the following internal standards: C17 Lactosyl(beta) ceramide (D18:1/17:0, 20 ppb), C17 Glucosyl(beta) ceramide (D18:1/17:0, 20 ppb), C17 ceramide (D18:1/17:0, 20 ppb), C16 ceramide-d7 (d18:1-d7/16:0, 16,57 ppb), C18 ceramide-d7 (d18:1-d7/18:0, 8.75 ppb), C24 ceramide-d7 (d18:1-d7/24:0, 20 ppb), and C24:1 ceramide-d7 (d18:1-d7/24:1(15Z), 9,96 ppb). The samples were the vortexed briefly and left on ice for 30 minutes. The samples were then centrifuged (9400g, 5 min, 4 °C) and then 60 µL of the bottom layer was transfer to a clean mass spectrometry vial (2 mL). The samples were then stored at –80 °C. The samples for this experiments were the same extracts that the Cer data was acquired from and had SM(18:1/17:0) spiked in prior to acquisition.
Processing Storage Conditions:	-80℃
Extract Storage:	-80℃

Combined analysis:

Analysis ID	AN002737
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Waters Acquity UHPLC UNSPSC 41115709
Column	Waters BEH C18 (00mm x 2.1mm,1.7um )
MS Type	ESI
MS instrument type	QTOF
MS instrument name	Bruker impact II UHPLC-QTOF system
Ion Mode	POSITIVE
Units	ng/ml

Chromatography:

Chromatography ID:	CH002023
Chromatography Summary:	Chromatographic separation was performed on an ACQUITY UHPLC BEH C18 column (2.1 mm × 100 mm, particle size 1.7 µm, Waters, Milford, MA, USA). The flow rate was set at 0.4 ml/min throughout the run with an injection volume of 1 µL. The following solvents were used for the gradient elution: Solvent A was H2O with 1% NH4Ac (1M) and HCOOH (0.1%) added. Solvent B was a mixture of ACN: IPA (1:1 v/v) with 1% NH4Ac (1M) and HCOOH (0.1%) added. The gradient was programmed as follows: 0 to 2 min 35-80% B, 2 to 7 min 80-100 % B, 7 to 14 min 100% B. The column was equilibrated with a 7min period of 35 % B prior to the next run.
Instrument Name:	Waters Acquity UHPLC UNSPSC 41115709
Column Name:	Waters BEH C18 (00mm x 2.1mm,1.7um )
Flow Gradient:	The gradient was programmed as follows: 0 to 2 min 35-80% B, 2 to 7 min 80-100 % B, 7 to 14 min 100% B. The column was equilibrated with a 7min period of 35 % B prior to the next run.
Flow Rate:	0.4ml/min
Solvent A:	100% water; 0.1% formic acid; 1% ammonium acetate
Solvent B:	50% acetonitrile/50% isopropanol; 0.1% formic acid; 1% ammonium acetate
Chromatography Type:	Reversed phase

MS:

MS ID:	MS002534
Analysis ID:	AN002737
Instrument Name:	Bruker impact II UHPLC-QTOF system
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	The SM results for UGCG-silenced Th17 cells data was acquired on a UHPLC-QTOF system from Bruker (Bruker, Billerica, MA, USA) combining an Elute UHPLC binary pump and an Impact II system QTOF system. The samples for this experiments were the same extracts that the Cer data was acquired from and had SM(18:1/17:0) spiked in prior to acquisition. The data was acquired using the Hystar suite of software. MZmine 2 was used for all the untargeted data processing.
Ion Mode:	POSITIVE