Homo sapiens
Protocols: Autologous memory CD4pos T-cells from Active or Remission RA were FACS-sorted from PBMCs based on their co-expression of CD3, CD4 and CD45RO. We used CD3 activating antibody to mimic TCR engagement. ST-DC DC2 or DC3 subsets and T-cells were sorted into FACS tubes containing complete RPMI1640 medium (10% FCS, penicillin/streptomycin 100U/mL, and 2mM Glutamax). Co-culture was set up when synovial biopsy yielded sufficient numbers of cells at least in one ST-DC subset (minimal 200 cells). Cells were co-cultured at a 1:5 ratio for each DC subset (200-1000 cells) plus memory T-cell (1000-4000 cells) in 200l of X-VIVO 15 Serum-free Cell Medium (BE02-060F, Lonza) supplemented with 2% human serum (H3667, Sigma) or in complete RPMI1640 in a 96-well round-bottom cell-culture plate (3799 – SLS, Corning). After 5 days in culture, the changes in T-cell phenotype were investigated using scRNAseq (BD Rhapsody Immune Response Panel, described in the section below). Cells for sequencing were isolated from culture wells by gentle pipetting prior to downstream preparation. Cells from from each culture condition were labelled with unique sample identifier tags using the BD Hu Single-Cell Sample Multiplexing Kit (633781/BD Bioscience) according to the manufacturer's protocol:The following sample Tags were used:Sample Tag 1— ATTCAAGGGCAGCCGCGTCACGATTGGATACGACTGTTGGACCGGSample Tag 2—TGGATGGGATAAGTGCGTGATGGACCGAAGGGACCTCGTGGCCGGSample Tag 3—CGGCTCGTGCTGCGTCGTCTCAAGTCCAGAAACTCCGTGTATCCTSample Tag 4—ATTGGGAGGCTTTCGTACCGCTGCCGCCACCAGGTGATACCCGCTSample Tag 5—CTCCCTGGTGTTCAATACCCGATGTGGTGGGCAGAATGTGGCTGGSample Tag 6—TTACCCGCAGGAAGACGTATACCCCTCGTGCCAGGCGACCAATGCSample Tag 7—TGTCTACGTCGGACCGCAAGAAGTGAGTCAGAGGCTGCACGCTGTSample Tag 8—CCCCACCAGGTTGCTTTGTCGGACGAGCCCGCACAGCGCTAGGATSample Tag 9—GTGATCCGCGCAGGCACACATACCGACTCAGATGGGTTGTCCAGGSample Tag 10—GCAGCCGGCGTCGTACGAGGCACAGCGGAGACTAGATGAGGCCCCSample Tag 11—CGCGTCCAATTTCCGAAGCCCCGCCCTAGGAGTTCCCCTGCGTGCSample Tag 12—GCCCATTCATTGCACCCGCCAGTGATCGACCCTAGTGGAGCTAAGCells were then loaded onto the scRNA-seq BD Rhapsody Cartridge using the BD Rhapsody Cartridge Reagent Kit (633731) according to the manufacturer's protocol. Single-cell cDNA was prepared using the BD Rhapsody cDNA Kit (633773). cDNA preparation was followed by single-cell mRNA and Tag library preparation using BD Rhapsody Targeted mRNA and the Tag Amplification Kit (633774) primers for the BD Rhapsody Immune Response Panel (399 genes: 633750).
ENA first public
2024-12-09
INSDC center name
School of Infection & Immunity, University of Glasgow, United Kingdom
INSDC status
public
Submitter Id
E-MTAB-14191_2:Library 1-2
broker name
ArrayExpress
cell_type
cultured ST DC and PB T-Cell
collection date
not collected
common name
human
developmental stage
adult
disease
rheumatoid arthritis
geographic location (country and/or sea)
not collected
isolate
not applicable
multiplexing
Multiplexed as in attached metadata
organism part
blood
sample name
E-MTAB-14191_2:Library 1-2
scientific_name
Homo sapiens