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Sample GSM2971182

Query DataSets for GSM2971182

Status

Public on Sep 26, 2018

Title

SLE267-Tfh

Sample type

SRA

Source name

whole blood

Organism

Homo sapiens

Characteristics

tissue: whole blood
subject id: SLE267
diagnosis: Systemic Lupus Erythematosus (SLE)
cell population (pd1pos=cxcr3+ pd1hi cd4+ t): Tfh
treatment: N/A
activation: N/A
project: 2

Treatment protocol

Cell isolation: For pDC isolation, the total dendritic cell fraction was obtained from healthy buffy coats by magnetic cell sorting with pan-DC Enrichment Kit (Stem Cell Technology). Highly pure (>99%) pDCs (Lin- HLADR+ CD11c- CD123+) were then isolated from this fraction by FACS sorting. Fresh peripheral blood naïve CD4 T cells (>99% pure) were isolated using Total CD4 T cell enrichment kit (Stem Cell Technology) followed by cell sorting as CD4+ CD45RA+ CD45RO- cells. Naïve CD4+ T cells where labeled with 5 μM carboxy-fluorescein diacetate succinimidyl ester (CFSE; Thermo Fisher) following the manufacturer’s instructions. Were described, primed (CFSElow) CD4+ T cells were sorted from pDC/CD4 T cell co-cultures at day 6. 7ADD and anti-CD123 antibody were used to remove dead cells and contaminating pDCs, respectively (Extended Data Fig. 2a). For B cell isolation, positively selected CD19+ B cells (Stem Cell Technology) were stained with anti-IgD APC, anti-CD27 PE and anti-CD19 FITC and then sorted as IgD+CD27−CD19+ cells (naïve) or IgD-CD27+CD19+ cells (memory). For blood CD4+ T cell subset sorting, frozen PBMCs from SLE patients were stained with anti-CD4 PE-Cy7, anti-CXCR5 AlexaFluor 647, anti-CD45RA APC-H7, anti-PD1 Brilliant Violet 421, anti-CD3 V500 and anti-CXCR3 Brilliant Violet 785. Then, CXCR3+ PD1low CD4+ T cells, CXCR3- PD1hi CD4+ T cells, CXCR3+ PD1hi CD4+ T cells populations were sorted from the CD3+ CD4+ CD45RA- CXCR5- cells fraction. CD3+ CD4+ CD45RA− CXCR5+ CD4+ T cells (Tfh cells) were also sorted for comparison. CD4 T cell differentiation, activation and analysis: 12 x 104 freshly sorted allogeneic naïve CD4+ T cells were co-cultured with activated pDCs (DC/T cell ratio of 1:6) in round-bottom 96-wells culture plates for 6 days. Were described, MitoTempo (MT – 50 μM – SantaCruz Biotechnology) or anti-PD1 antibody (clone EH12.2H7 – 10 μg/ml - Biolegend) were added during the co-culture. As a control, 12 x 104 naïve CD4+ T cells were activated with 1 μl of Dynabeads human T cell activator CD3/CD28 (Thermo Fisher) for 6 days (referred in the text as “Th0”). For intracellular cytokine staining, primed CD4+ T cells were re-stimulated with 50 ng/ml PMA, 2 μg/ml Ionomycin and GolgiPlug (BD Biosciences) for 5 h. Cells were then stained with a combination of anti-IL10 and IFNγ mAbs (BD Biosciences) with Cytofix/Cytoperm Fixation and Permeabilization Solution (BD Biosciences) following the manufacturer’s instructions. For re-stimulation experiments, 5 x 104 primed (CFSElow) CD4+ T cells or sorted SLE patient CD4+ T cells were re-stimulated with 10 μg/ml of plate-bound anti-CD3 (OKT3 - Biolegend) and 2 μg/ml of soluble anti-CD28 mAb (Biolegend) for 24 h. Cytokine levels in the corresponding supernatants were measured with Flex Set Kit (BD Biosciences). For HIF1α intracellular staining, CD4+ T cells were stained with anti-HIF-1α antibody (Biolegend) with Foxp3 fix/perm buffer set (Biolegend) following the manufacturer’s instructions. Dead cells were excluded from the analysis by labeling with LIVE/DEAD fixable Aqua (Thermo Fisher) prior to fixation/permeabilization. Proliferation assay: CD4 T cell proliferation was measured by resazurin reduction, as previously described3. Briefly, primed (CFSElow) CD4+ T cells were seeded at 5 × 105 cells and re-stimulated with 10 μg/ml of plate-bound anti-CD3 (OKT3) and 2 μg/ml of soluble anti-CD28 mAbs. After incubation for 20 h, medium supplemented with 40 μM resazurin was added for an additional 4 hr. Resazurin reduction to resorufin was measured fluorometrically using a SpectraMax M5 (Molecular Devices). Results obtained were expressed in fluorescence arbitrary units (AU). Alternatively, proliferation was assessed by flow cytometry using the Click-IT EdU Proliferation Kit, according to the manufacturer’s instructions (Thermo Fisher). In vitro Tfh cell generation: Human Tfh cells were generated as previously described. Briefly, after overnight stimulation of naive CD4+ T cells with Dynabeads human T cell activator CD3/CD28 (Thermo Fisher) in complete RPMI medium, cells were transferred to 96-well plates coated with anti-CD3 and supplemented with 2 μg/ml of soluble anti-CD28 mAb (Biolegend), human recombinant IL-23 (25 ng/ml) and human recombinant TGFβ (5 ng/ml). After 4 d cells were collected and used for analysis.

Growth protocol

Blood samples were obtained from patients fulfilling the diagnosis of SLE according to the criteria established by the American College of Rheumatology. Healthy pediatric controls were children visiting the clinic either for reasons not related to autoimmunity or for surgery not associated with any inflammatory diseases

Extracted molecule

total RNA

Extraction protocol

Total RNA was isolated using the RNeasy kit (Qiagen)
Total RNA was converted to cDNA using 20 ng RNA as input for the Ovation RNA-Seq System V2, and barcoded Illumina library constructs were produced using Ovation Ultralow Library System V2 (NuGEN Technologies) following the manufacturer’s instructions. Library fragment size and molar concentration were determined using Agilent High Sensitivity DNA Chip on 2100 Bioanalyzer, and quantitative PCR data were obtained by the KAPA BioSystems Library Quantification qPCR Kit for Illumina Platform on a Viia7 Real-Time PCR System (Thermo Fisher Scientific). RNA-seq libraries were sequenced on the Illumina HiSeq 2500 sequencing system using SBS v3 chemistry and two high-output flow cells targeting 70–80 million 2 × 75 paired-end reads per library.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

project2.csv
cDNA3645

Data processing

Sequences were aligned with HISAT2, duplicates removed with Samtools, and counts generated with HTSeq using the annotations from Gencode V20. Genes identified as globins, ribosomal RNAs, and pseudogenes were removed.
Genome_build: GRCh37/hg19
Supplementary_files_format_and_content: csv; raw counts

Submission date

Jan 30, 2018

Last update date

Mar 16, 2023

Contact name

Nicole Baldwin

E-mail(s)

[email protected]

Organization name

Baylor Research Institute

Street address

3434 Live Oak St