Easysep cd4 enrichment kit

Healthy donor PBMCs were obtained from the Etablissement Français du Sang. PBMCs were typed based on the expression or absence of HLA-A2/A28 molecules, as assessed by anti-HLA-A2/A28 antibody (OneLambda) staining evaluated by flow cytometry using the BD LSRFortessa^TM X-20 analyzer. CD4+ T cells were enriched from HLA-A2- donor PBMCs using an EasySep CD4+ Enrichment kit (Stem Cell®). Naive regulatory T cells (nTregs), which were defined as CD4+ CD25^Hi CD127− CD45RA+ CD45RO−, were sorted using a FACS Aria II (BD Biosciences). In parallel, we sorted CD4+ CD45RA+ CD25− Tconv cells from the same donor as controls. The sorting purity was checked with flow cytometry and always demonstrated a purity greater than 98%. Sorted T cells were stimulated with Dynabeads^TM Human T-Activator CD3/CD28 (Thermo Fisher Scientific) (ratio 1:1) in X-VIVO® 20 medium containing 10% human serum AB (Biowest) and 1000 UI/mL human IL-2 (Proleukin, Novartis). HLA-typed CD3-depleted splenocytes were isolated from spleens collected from deceased organ donors through collaboration with the Regional Histocompatibility Laboratory and National Biomedicine Agency.

PBMCs were isolated from reduction chambers obtained from Vitalant Research Institute and Stanford Blood Bank using Ficoll-Hypaque density gradients, and then cultured in R10. For sorting experiments, CD4+ T cells were purified by negative selection using the EasySep CD4 enrichment kit (Stem Cell Technologies), and further enriched for memory cells by depletion of naïve T cells using CD45RA beads (Miltenyi Biotec), prior to lectin staining and sorting as described further below. Where indicated, PBMCs were first stimulated for 3 days with 5 µg/ml PHA in the presence of 10 IU/ml IL-2 prior to CyTOF-Lec analysis.

Blood samples were obtained from eight healthy adults, aged from 22 to 46 years. Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque PLUS (GE healthcare, Buckingham, UK) density gradient centrifugation. CD4⁺ T cells were isolated from PBMCs using the negative selection EasySep® CD4⁺ enrichment kit (StemCell Technologies, Meylan, France) according to the manufacturer’s instructions. DNA was isolated from live PBMCs using Qiagen DNeasy blood and tissue kit.
Samples used for RNA extraction analysis were obtained in accordance with the commercial vendor’s approved institutional review board protocols. The sample used for ATAC-seq and H3K27ac ChM-seq were obtained from NHSBT Cambridgeshire. All research use was approved by the Research Ethics Committee (reference number: 15/NW/0282).