Unknown,Transcriptomics,Genomics,Proteomics

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Single cell RNAseq of peripheral CD4+ T cells in the context SARS-CoV-2 infection

ABSTRACT: CD4+ T cells play a fundamental role in host defenses against viruses by orchestrating B cell development and/or by directly targeting pathogens. This dataset aimed at understanding the molecular profile of CD4+ T cells in the context of an acute and severe SARS-CoV-2 infection. The analyzed patient was recruited at Henri Mondor University Hospital (AP-HP, Paris France) in March 2020, and required oxygen as treatment. Peripheral (CD14-CD56-CD123-CD19-CD3+CD4+CD45RA-PD-1high) T cells were FACS-sorted using a Sony MA900 in PBS/0.08% FCS. 1.105 cells were obtained and 20000 were loaded in the 10x Chromium Controller to generate single-cell gel-beads in emulsion. The scRNA-seq libraries were generated using the Chromium Next GEM Single Cell V(D)J Reagent Kit v.1.1 with Feature Barcoding (10x Genomics) according to the manufacturer’s protocol. PBMCs were initially isolated from venous blood samples via standard density gradient centrifugation and used after cryopreservation at -150°C. Cells were thawed using RPMI-1640 (Gibco) 10% FBS, washed twice and incubated with fluorochrome-conjugated antibody cocktail and viable cells were identified using a LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific) incubated with conjugated antibodies used for cell sorting (CD3/CD4/CD14/CD19/CD45/CD56/CD123/PD-1) as well as a panel of barcoded TotalSeqC® antibodies (CD11c/CD21/CD27/CD38/CD49d/CD69/CD71/CD73/CD95/CD103/CD138/CD305/CD307d/CD307e/CXCR3/CXCR4/CXCR5/HLA-DR/ICOS/ITB7/LAG-3/NKG2D/TIM3). A single sort was performed for this patient and 5’-transcriptomic, VDJ and ADT libraries were sequenced .

INSTRUMENT(S): Illumina NovaSeq 6000, Sony MA900

ORGANISM(S): Homo sapiens

SUBMITTER: Manfroi Benoît

PROVIDER: E-MTAB-13321 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Memory B cells play a fundamental role in host defences against viruses. This dataset aimed at understanding their maturation and stability in the context of SARS-CoV-2 infection and consist of a longitudinal single-cell and repertoire profiling of the B cell response up to six months in four severe COVID-19 patients. All four patients were recruited at Henri Mondor University Hospital (AP-HP, Paris France), between March and May 2020, and required oxygen as treatment. Clinical and biological characteristics of these patients are summarized in the Patient_information.csv file. Peripheral (CD3-CD14-CD15-CD56-CD19+IgD-) B cells were FACS-sorted (MA900, Sony) in PBS/0.08% FCS from 4 patients (S-CoV) at baseline (M0) and 6 months (M6). 5x104 to 10x105 cells were obtained for each subset and 20000 were loaded in the 10x Chromium Controller to generate single-cell gel-beads in emulsion. The scRNA-seq libraries were generated using the Chromium Next GEM Single Cell V(D)J Reagent Kit v.1.1 with Feature Barcoding (10x Genomics) according to the manufacturer’s protocol. PBMCs were initially isolated from venous blood samples via standard density gradient centrifugation and used after cryopreservation at -150°C. Cells were thawed using RPMI-1640 (Gibco) 10% FBS, washed twice and incubated with 10µg of the SARS-CoV-2 his tagged spike protein in 100µL of PBS (Gibco) 2% FBS during 20 minutes on ice. Cells were washed and resuspended in the same conditions, then fluorochrome-conjugated antibody cocktail including the 2 anti-His was added at pre-titrated concentrations for 20 min at 4°C and viable cells were identified using a LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific) incubated with conjugated antibodies used for cell sorting (CD3/CD14/CD15/CD56/CD19/IgD) as well as a panel of barcoded TotalSeqC® and homemade anti-His antibodies (see feature_reference.csv.gz files). Three distinct sorts were performed for each donor: two at the M0 time-point (M0_Sort1 and M0_Sort2) and one at the M6 time-point (M6_Sort1). His-tagged-Spike + barcoded-anti-his staining was only included in the last two sorts (M0_Sort2 and M6_Sort1). For these two sorts it should additionally be noted that 5’-transcriptomic and ADT libraries were sequenced in two separate runs (Run1 and Run2) to achieve sufficient sequencing depth for both libraries. Both runs were pooled at the Cell Ranger analysis step.

Project description:Memory B cells play a fundamental role in host defenses against viruses. This dataset aimed at understanding the recruitment and remodeling of the memory B cell repertoire in the context of BA.1-breakthrough infection in BNT162b2 mRNA vaccinated individuals. All four donors enrolled in the study had received a booster (3rd dose) of BNT162b2 mRNA vaccine and had no history of prior SARS-CoV-2 infection. All four experienced a documented breakthrough infection between 12/24/2021 and 01/30/2022 when BA.1 was responsible for > 85% of SARS-CoV-2 infections in France. All donors were sampled at Henri Mondor University Hospital (AP-HP, Paris France), and samples used for scRNA-seq were collected shortly after breakthrough infection (PO_M0 samples; between day 7 and day 18) and 5 to 6 months after infection (PO_M6 samples; between day 152 and day 173). Clinical and biological characteristics of these patients are summarized in the Patient_information.csv file. For each sample, an initial pool of 50.000 total peripheral CD3-CD14-CD56- CD19+ IgD- cells was always sorted and afterward, to enrich for cells of interest, only CD19+CD38low antibody secreting cells (ASCs), CD19hi IgD+ cells and SARS-CoV-2 Spike/RBD PE/tetramer positive B cells were sorted, leading to approximately 55000-60000 total sorted cells per sample. Sorted cells were then counted and up to 20 000 cells were loaded in the 10x Chromium Controller to generate single-cell gel-beads in emulsion. The scRNA-seq libraries for gene expression (mRNA), ADT and VDJ BCR libraries were generated using Chromium Next GEM Single Cell V(D)J Reagent Kit v.1.1 with Feature Barcoding (10x Genomics) according to the manufacturer’s protocol. PBMCs were initially isolated from venous blood samples via standard density gradient centrifugation and used after cryopreservation at -150°C. PBMCs were thawed using RPMI-1640 (Gibco) 10% FBS, washed twice and approximately 15x106 cells were then resuspended in 100µL PBS 2%FBS and incubated for 40 minutes at 4°C with a decoy tetramer (biotinylated Bovine Serum albumin coupled with BV785 streptavidin) and Hu-1 Spike, BA.1 Spike, Hu-1 RBD and BA.1 RBD tetramers (constructed using PE-labelled TotalSeqC® streptavidin with different barcodes for each individual antigens (see feature_reference.csv.gz files).). Cells were washed, resuspended in 100µL PBS 2%FBS and stained with a cocktail of fluorochrome conjugated (CD3, CD14 both APC-H7 at 1:100 each; CD15 and CD56 BV785 at 1:100 each, CD19 PECF594 at 1:100, IgD FITC at 1:100, CD38 PercP-Cy5.5 at 1:100) and TotalSeqC® (CD38, CD27, CD71, CD21, CD11c, CD39, FCRL5, CD95 all at 1:40 (all obtained from BioLegend)) antibodies for 40 minutes on ice. Viable cells were identified using a LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific, 1:200) incubated with conjugated antibodies. Two distinct sorts were performed for each donor: one at the early time-point (PO_M0) and one at the 6 months’ time-point (PO_M6).

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Single cell RNAseq of peripheral CD4+ T cells in the context SARS-CoV-2 infection

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