Summary of Study ST001675

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	ST001675
Study Title	Quantitative measurements of ceramides and glycosphingolipids in Th17 and iTreg cells (part-II)
Study Type	MS: Targeted analysis
Study Summary	Part 2/5: It includes quantitative targeted measurements of sphingolipids (ceramides and glycosphingolipids) in Th17, iTreg, and their paired controls (Th0 cells).
Institute	University of Turku
Department	Systems Medicine, Turku Bioscience
Laboratory	Metabolomics
Last Name	Sen
First Name	Partho
Address	Tykistökatu 6B, BioCity, 5th Floor
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

Select appropriate tab below to view additional metadata details:

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:	SU001752
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	Cell Type	Treatment	Types
SA154498	iTreg_D5	iTreg	Differentiated	Test
SA154499	iTreg_D1	iTreg	Differentiated	Test
SA154500	iTreg_D2	iTreg	Differentiated	Test
SA154501	iTreg_D3	iTreg	Differentiated	Test
SA154502	iTreg_D4	iTreg	Differentiated	Test
SA154489	Th0(iTreg)_D1	Th0(iTreg)	Activated	Control
SA154490	Th0(iTreg)_D5	Th0(iTreg)	Activated	Control
SA154491	Th0(iTreg)_D2	Th0(iTreg)	Activated	Control
SA154492	Th0(iTreg)_D3	Th0(iTreg)	Activated	Control
SA154493	Th0(iTreg)_D4	Th0(iTreg)	Activated	Control
SA154485	Th0(Th17)_D1	Th0(Th17)	Activated	Control
SA154486	Th0(Th17)_D3	Th0(Th17)	Activated	Control
SA154487	Th0(Th17)_D2	Th0(Th17)	Activated	Control
SA154488	Th0(Th17)_D4	Th0(Th17)	Activated	Control
SA154494	Th17_D4	Th17	Differentiated	Test
SA154495	Th17_D1	Th17	Differentiated	Test
SA154496	Th17_D3	Th17	Differentiated	Test
SA154497	Th17_D2	Th17	Differentiated	Test

Showing results 1 to 18 of 18

Showing results 1 to 18 of 18

Collection:

Collection ID:	CO001745
Collection Summary:	CD4+ T-cells were isolated from human umbilical cord blood as described previously (Khan et al., 2020; Tripathi et al., 2017; Ubaid et al., 2018). 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:	TR001765
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:

TR001765

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:	SP001758
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.
Processing Storage Conditions:	-80℃
Extract Storage:	-80℃

Combined analysis:

Analysis ID	AN002733
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	Triple quadrupole
MS instrument name	ABI Sciex 5500 QTrap
Ion Mode	POSITIVE
Units	ng/ml

Chromatography:

Chromatography ID:	CH002019
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:	MS002530
Analysis ID:	AN002733
Instrument Name:	ABI Sciex 5500 QTrap
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	The ceramides were quantified using a targeted multiple reaction monitoring (MRM) method using UHPLC as a separation technique. The LC separation was based on the global lipidomics method previously described 71. Briefly, the UHPLC was a Exion AD (Sciex) integrated system. The samples were held in a cool box at 15 °C prior to the analysis. The needle was washed with both a 10% DCM in MeOH and ACN: MeOH: IPA: H2O (1:1:1:1 v/v/v/v) with 1% HCOOH for a total of 7.5 seconds each. The solvents were delivered using a quaternary solvent and a column oven (set to 50 °C). The 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. The mass spectrometer was a Sciex 5500 QTRAP (Sciex) set in scheduled MRM mode. All lipids were identified for their fatty acid composition by MS/MS to confirm their exact identification, there was also a linear relationship between the increasing number of carbons in the lipid chain and its corresponding retention time. Due to the isobaric nature of sugars we were unable to differentiate Glc and Glc head groups. All data were integrated using the quantitation tool in MultiQuant (3.0.3), all peaks were manually checked. Any analytes which were over the concentration of the standard curve were diluted (1:25) with the same extraction solvent minus the internal standards. The quantification was performed using class-based internal standards and in the case of those ceramide species without an authentic standard in the standard curve mix, we used the closest related structure. The standard curve mixture contained: Glucosyl (beta) C12 ceramide, Lactosyl (beta)) C12 ceramide, C18 ceramide (D18:1/18:1), C18:1 dihydroceramide (d18:0/18:1(9Z)) and was run at the following levels (all in ppb): 100, 80, 60, 50, 40, 30, 20, 10 for the C12 standards and 10, 8, 6, 5, 4, 3, 2,1 for all C18 standards.
Ion Mode:	POSITIVE